Albumin fusion proteins

ABSTRACT

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disordrs or conditions using albumin fusion proteins of the invention.

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) based on the following U.S. provisional applications: No.60/229,358 filed on Apr. 12, 2000; No. 60/199,384 filed on Apr. 25,2000; and No. 60/256,931 filed on Dec. 21, 2000. Each of the provisionalapplications is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The invention relates generally to Therapeutic proteins(including, but not limited to, a polypeptide, antibody, or peptide, orfragments and variants thereof) fused to albumin or fragments orvariants of albumin. The invention further relates to Therapeuticproteins (including, but not limited to, a polypeptide, antibody, orpeptide, or fragments and variants thereof) fused to albumin orfragments or variants of albumin, that exhibit extended shelf-lifeand/or extended or therapeutic activity in solution. These fusionproteins are herein collectively referred to as “albumin fusion proteinsof the invention.” The invention encompasses therapeutic albumin fusionproteins, compositions, pharmaceutical compositions, formulations andkits. Nucleic acid molecules encoding the albumin fusion proteins of theinvention are also encompassed by the invention, as are vectorscontaining these nucleic acids, host cells transformed with thesenucleic acids vectors, and methods of making the albumin fusion proteinsof the invention using these nucleic acids, vectors, and/or host cells.

[0003] The invention is also directed to methods of in vitro stabilizinga Therapeutic protein via fusion or conjugation of the Therapeuticprotein to albumin or fragments or variants of albumin.

[0004] Human serum albumin (HSA, or HA), a protein of 585 amino acids inits mature form (as shown in FIG. 15 or in SEQ ID NO:18), is responsiblefor a significant proportion of the osmotic pressure of serum and alsofunctions as a carrier of endogenous and exogenous ligands. At present,HA for clinical use is produced by extraction from human blood. Theproduction of recombinant HA (rHA) in microorganisms has been disclosedin EP 330 451 and EP 361 991.

[0005] The role of albumin as a carrier molecule and its inert natureare desirable properties for use as a carrier and transporter ofpolypeptides in vivo. The use of albumin as a component of an albuminfusion protein as a carrier for various proteins has been suggested inWO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminalfragments of HA for fusions to polypeptides has also been proposed (EP399 666). Fusion of albumin to the Therapeutic protein may be achievedby genetic manipulation, such that the DNA coding for HA, or a fragmentthereof, is joined to the DNA coding for the Therapeutic protein. Asuitable host is then transformed or transfected with the fusednucleotide sequences, so arranged on a suitable plasmid as to express afusion polypeptide. The expression may be effected in vitro from, forexample, prokaryotic or eukaryotic cells, or in vivo e.g. from atransgenic organism.

[0006] Therapeutic proteins in their native state or when recombinantlyproduced, such as interferons and growth hormones, are typically labilemolecules exhibiting short shelf-lives, particularly when formulated inaqueous solutions. The instability in these molecules when formulatedfor administration dictates that many of the molecules must belyophilized and refrigerated at all times during storage, therebyrendering the molecules difficult to transport and/or store. Storageproblems are particularly acute when pharmaceutical formulations must bestored and dispensed outside of the hospital environment. Many proteinand peptide drugs also require the addition of high concentrations ofother protein such as albumin to reduce or prevent loss of protein dueto binding to the container. This is a major concern with respect toproteins such as IFN. For this reason, many Therapeutic proteins areformulated in combination with large proportion of albumin carriermolecule (100-1000 fold excess), though this is an undesirable andexpensive feature of the formulation.

[0007] Few practical solutions to the storage problems of labile proteinmolecules have been proposed. Accordingly, there is a need forstabilized, long lasting formulations of proteinaceous therapeuticmolecules that are easily dispensed, preferably with a simpleformulation requiring minimal post-storage manipulation.

SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, on the discovery thatTherapeutic proteins may be stabilized to extend the shelf-life, and/orto retain the Therapeutic protein's activity for extended periods oftime in solution, in vitro and/or in vivo, by genetically or chemicallyfusing or conjugating the Therapeutic protein to albumin or a fragment(portion) or variant of albumin, that is sufficient to stabilize theprotein and/or its activity. In addition it has been determined that theuse of albumin-fusion proteins or albumin conjugated proteins may reducethe need to formulate protein solutions with large excesses of carrierproteins (such as albumin, unfused) to prevent loss of Therapeuticproteins due to factors such as binding to the container.

[0009] The present invention encompasses albumin fusion proteinscomprising a Therapeutic protein (e.g., a polypeptide, antibody, orpeptide, or fragments and variants thereof) fused to albumin or afragment (portion) or variant of albumin. The present invention alsoencompasses albumin fusion proteins comprising a Therapeutic protein(e.g., a polypeptide, antibody, or peptide, or fragments and variantsthereof) fused to albumin or a fragment (portion) or variant of albumin,that is sufficient to prolong the shelf life of the Therapeutic protein,and/or stabilize the Therapeutic protein and/or its activity in solution(or in a pharmaceutical composition) in vitro and/or in vivo. Nucleicacid molecules encoding the albumin fusion proteins of the invention arealso encompassed by the invention, as are vectors containing thesenucleic acids, host cells transformed with these nucleic acids vectors,and methods of making the albumin fusion proteins of the invention andusing these nucleic acids, vectors, and/or host cells.

[0010] The invention also encompasses pharmaceutical formulationscomprising an albumin fusion protein of the invention and apharmaceutically acceptable diluent or carrier. Such formulations may bein a kit or container. Such kit or container may be packaged withinstructions pertaining to the extended shelf life of the Therapeuticprotein. Such formulations may be used in methods of treating,preventing, ameliotationg or diagnosing a disease or disease symptom ina patient, preferably a mammal, most preferably a human, comprising thestep of administering the pharmaceutical formulation to the patient.

[0011] In other embodiments, the present invention encompasses methodsof preventing treating, or ameliorating a disease or disorder. Inpreferred embodiments, the present invention encompasses a method oftreating a disease or disorder listed in the “Preferred Indication Y”column of Table 1 comprising administering to a patient in which suchtreatment, prevention or amelioration is desired an albumin fusionprotein of the invention that comprises a Therapeutic protein portioncorresponding to a Therapeutic protein (or fragment or variant thereof)disclosed in the “Therapeutic Protein X” column of Table 1 (in the samerow as the disease or disorder to be treated is listed in the “PreferredIndication Y” column of Table 1) in an amount effective to treat preventor ameliorate the disease or disorder.

[0012] In another embodiment, the invention includes a method ofextending the shelf life of a Therapeutic protein (e.g., a polypeptide,antibody, or peptide, or fragments and variants thereof) comprising thestep of fusing or conjugating the Therapeutic protein to albumin or afragment (portion) or variant of albumin, that is sufficient to extendthe shelf-life of the Therapeutic protein. In a preferred embodiment,the Therapeutic protein used according to this method is fused to thealbumin, or the fragment or variant of albumin. In a most preferredembodiment, the Therapeutic protein used according to this method isfused to albumin, or a fragment or variant of albumin, via recombinantDNA technology or genetic engineering.

[0013] In another embodiment, the invention includes a method ofstabilizing a Therapeutic protein (e.g., a polypeptide, antibody, orpeptide, or fragments and variants thereof) in solution, comprising thestep of fusing or conjugating the Therapeutic protein to albumin or afragment (portion) or variant of albumin, that is sufficient tostabilize the Therapeutic protein. In a preferred embodiment, theTherapeutic protein used according to this method is fused to thealbumin, or the fragment or variant of albumin. In a most preferredembodiment, the Therapeutic protein used according to this method isfused to albumin, or a fragment or variant of albumin, via recombinantDNA technology or genetic engineering.

[0014] The present invention further includes transgenic organismsmodified to contain the nucleic acid molecules of the invention,preferably modified to express the albumin fusion proteins encoded bythe nucleic acid molecules.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 depicts the extended shelf-life of an HA fusion protein interms of the biological activity (Nb2 cell proliferation) of HA-hGHremaining after incubation in cell culture media for up to 5 weeks at37° C. Under these conditions, hGH has no observed activity by week 2.

[0016]FIG. 2 depicts the extended shelf-life of an HA fusion protein interms of the stable biological activity (Nb2 cell proliferation) ofHA-hGH remaining after incubation in cell culture media for up to 3weeks at 4, 37, or 50° C. Data is normalized to the biological activityof hGH at time zero.

[0017]FIGS. 3A and 3B compare the biological activity of HA-hGH with hGHin the Nb2 cell proliferation assay. FIG. 3A shows proliferation after24 hours of incubation with various concentrations of hGH or the albuminfusion protein, and FIG. 3B shows proliferation after 48 hours ofincubation with various concentrations of hGH or the albumin fusionprotein.

[0018]FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as thebase vector into which polynucleotides encoding the Therapeutic proteins(including polypeptides and fragments and variants thereof) may becloned to form HA-fusions. Plasmid Map key: PRB1p: PRB1 S. cerevisiaepromoter; FL: Fusion leader sequence; rHA: cDNA encoding HA; ADH1t: ADH1S. cerevisiae terminator; T3: T3 sequencing primer site; T7: T7sequencing primer site; Amp R: β-lactamase gene; ori: orgi n ofreplication. Please note that in the provisional applications to whichthis application claims priority, the plasmid in FIG. 4 was labeledpPPC0006, instead of pPPC0005. In addition the drawing of this plasmiddid not show certain pertinent restriction sites in this vector. Thus inthe present application, the drawing is labeled pPPC0005 and morerestriction sites of the same vector are shown.

[0019]FIG. 5 compares the recovery of vial-stored HA-IFN solutions ofvarious concentrations with a stock solution after 48 or 72 hours ofstorage.

[0020]FIG. 6 compares the activity of an HA-α-IFN fusion protein afteradministration to monkeys via IV or SC.

[0021]FIG. 7 describes the bioavailability and stability of an HA-α-IFNfusion protein.

[0022]FIG. 8 is a map of an expression vector for the production ofHA-α-IFN.

[0023]FIG. 9 shows the location of loops in HA.

[0024]FIG. 10 is an example of the modification of an HA loop.

[0025]FIG. 11 is a representation of the HA loops.

[0026]FIG. 12 shows the HA loop IV.

[0027]FIG. 13 shows the tertiary structure of HA.

[0028]FIG. 14 shows an example of a scFv-HA fusion FIG. 15 shows theamino acid sequence of the mature form of human albumin (SEQ ID NO:18)and a polynucleotide encoding it (SEQ ID NO:17).

DETAILED DESCRIPTION

[0029] As described above, the present invention is based, in part, onthe discovery that a Therapeutic protein (e.g., a polypeptide, antibody,or peptide, or fragments and variants thereof) may be stabilized toextend the shelf-life and/or retain the Therapeutic protein's activityfor extended periods of time in solution (or in a pharmaceuticalcomposition) in vitro and/or in vivo, by genetically fusing orchemically conjugating the Therapeutic protein, polypeptide or peptideto all or a portion of albumin sufficient to stabilize the protein andits activity.

[0030] The present invention relates generally to albumin fusionproteins and methods of treating, preventing, or ameliorating diseasesor disorders. As used herein, “albumin fusion protein” refers to aprotein formed by the fusion of at least one molecule of albumin (or afragment or variant thereof) to at least one molecule of a Therapeuticprotein (or fragment or variant thereof). An albumin fusion protein ofthe invention comprises at least a fragment or variant of a Therapeuticprotein and at least a fragment or variant of human serum albumin, whichare associated with one another, preferably by genetic fusion (i.e., thealbumin fusion protein is generated by translation of a nucleic acid inwhich a polynucleotide encoding all or a portion of a Therapeuticprotein is joined in-frame with a polynucleotide encoding all or aportion of albumin) or chemical conjugation to one another. TheTherapeutic protein and albumin protein, once part of the albumin fusionprotein, may be referred to as a “portion”, “region” or “moiety” of thealbumin fusion protein (e.g., a “Therapeutic protein portion” or an“albumin protein portion”).

[0031] In one embodiment, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a Therapeuticprotein (e.g., as described in Table 1) and a serum albumin protein. Inother embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment of a Therapeutic protein and a serumalbumin protein. In other embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, abiologically active and/or therapeutically active variant of aTherapeutic protein and a serum albumin protein. In preferredembodiments, the serum albumin protein component of the albumin fusionprotein is the mature portion of serum albumin.

[0032] In further embodiments, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a Therapeuticprotein, and a biologically active and/or therapeutically activefragment of serum albumin. In further embodiments, the inventionprovides an albumin fusion protein comprising, or alternativelyconsisting of, a Therapeutic protein and a biologically active and/ortherapeutically active variant of serum albumin. In preferredembodiments, the Therapeutic protein portion of the albumin fusionprotein is the mature portion of the Therapeutic protein. In a furtherpreferred embodiment, the Therapeutic protein portion of the albuminfusion protein is the extracellular soluble domain of the Therapeuticprotein. In an alternative embodiment, the Therapeutic protein portionof the albumin fusion protein is the active form of the Therapeuticprotien.

[0033] In further embodiments, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a biologicallyactive and/or therapeutically active fragment or variant of aTherapeutic protein and a biologically active and/or therapeuticallyactive fragment or variant of serum albumin. In preferred embodiments,the invention provides an albumin fusion protein comprising, oralternatively consisting of, the mature portion of a Therapeutic proteinand the mature portion of serum albumin.

[0034] Therapeutic Proteins

[0035] As stated above, an albumin fusion protein of the inventioncomprises at least a fragment or variant of a Therapeutic protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, preferably by genetic fusion or chemical conjugation.

[0036] As used herein, “Therapeutic protein” refers to proteins,polypeptides, antibodies, peptides or fragments or variants thereof,having one or more therapeutic and/or biological activities. Therapeuticproteins encompassed by the invention include but are not limited to,proteins, polypeptides, peptides, antibodies, and biologics. (The termspeptides, proteins, and polypeptides are used interchangeably herein.)It is specifically contemplated that the term “Therapeutic protein”encompasses antibodies and fragments and variants thereof. Thus analbumin fusion protein of the invention may contain at least a fragmentor variant of a Therapeutic protein, and/or at least a fragment orvariant of an antibody. Additionally, the term “Therapeutic protein” mayrefer to the endogenous or naturally occurring correlate of aTherapeutic protein.

[0037] By a polypeptide displaying a “therapeutic activity” or a proteinthat is “therapeutically active” is meant a polypeptide that possessesone or more known biological and/or therapeutic activities associatedwith a Therapeutic protein such as one or more of the Therapeuticproteins described herein or otherwise known in the art. As anon-limiting example, a “Therapeutic protein” is a protein that isuseful to treat, prevent or ameliorate a disease, condition or disorder.As a non-limiting example, a “Therapeutic protein” may be one that bindsspecifically to a particular cell type (normal (e.g., lymphocytes) orabnormal e.g., (cancer cells)) and therefore may be used to target acompound (drug, or cytotoxic agent) to that cell type specifically.

[0038] In another non-limiting example, a “Therapeutic protein” is aprotein that has a biological activity, and in particular, a biologicalactivity that is useful for treating preventing or ameliorating adisease. A non-inclusive list of biological activities that may bepossessed by a Therapeutic protein includes, enhancing the immuneresponse, promoting angiogenesis, inhibiting angiogenesis, regulatinghematopoietic functions, stimulating nerve growth, enhancing an immuneresponse, inhibiting an immune response, or any one or more of thebiological activities described in the “Biological Activities” sectionbelow.

[0039] As used herein, “therapeutic activity” or “activity” may refer toan activity whose effect is consistent with a desirable therapeuticoutcome in humans, or to desired effects in non-human mammals or inother species or organisms. Therapeutic activity may be measured in vivoor in vitro. For example, a desirable effect may be assayed in cellculture. As an example, when hGH is the Therapeutic protein, the effectsof hGH on cell proliferation as described in Example 1 may be used asthe endpoint for which therapeutic activity is measured. Such in vitroor cell culture assays are commonly available for many Therapeuticproteins as described in the art.

[0040] Examples of useful assays for particular Therapeutic proteinsinclude, but are not limited to, GMCSF (Eaves, A. C. and Eaves C. J.,Erythropoiesis in culture. In: McCullock E A (edt) Cell culturetechniques—Clinics in hematology. W B Saunders, Eastbourne, pp 371-91(1984); Metcalf, D., International Journal of Cell Cloning 10: 116-25(1992); Testa, N. G., et al., Assays for hematopoietic growth factors.In: Balkwill F R (edt) Cytokines A practical Approach, pp 229-44; IRLPress Oxford 1991) EPO (bioassay: Kitamura et al., J. Cell. Physiol. 140p323 (1989)); Hirudin (platelet aggregation assay: Blood CoagulFibrinolysis 7(2):259-61 (1996)); IFNα (anti-viral assay: Rubinstein etal., J. Virol. 37(2):755-8 (1981); anti-proliferative assay: Gao Y, etal Mol Cell Biol. 19(11):7305-13 (1999); and bioassay: Czarniecki etal., J. Virol. 49 p490 (1984)); GCSF (bioassay: Shirafuji et al., Exp.Hematol. 17 p116 (1989); proliferation of murine NFS-60 cells (Weinsteinet al, Proc Natl Acad Sci 83:5010-4 (1986)); insulin (3H-glucose uptakeassay: Steppan et al., Nature 409(6818):307-12 (2001)); hGH (Ba/F3-hGHRproliferation assay: J Clin Endocrinol Metab 85(11):4274-9 (2000);International standard for growth hormone: Horm Res, 51 Suppl 1:7-12(1999)); factor X (factor X activity assay: Van Wijk et al. Thromb Res22:681-686 (1981)); factor VII (coagulation assay using prothrombinclotting time: Belaaouaj et al., J. Biol. Chem. 275:27123-8(2000);Diaz-Collier et al., Thromb Haemost 71:339-46 (1994)), or as shown inTable 1 in the “Exemplary Activity Assay” column.

[0041] Therapeutic proteins corresponding to a Therapeutic proteinportion of an albumin fusion protein of the invention, such as cellsurface and secretory proteins, are often modified by the attachment ofone or more oligosaccharide groups. The modification, referred to asglycosylation, can dramatically affect the physical properties ofproteins and can be important in protein stability, secretion, andlocalization. Glycosylation occurs at specific locations along thepolypeptide backbone. There are usually two major types ofgiycosylation: glycosylation characterized by O-linked oligosaccharides,which are attached to serine or threonine residues; and glycosylationcharacterized by N-linked oligosaccharides, which are attached toasparagine residues in an Asn-X-Ser/Thr sequence, where X can be anyamino acid except proline. N-acetylneuramic acid (also known as sialicacid) is usually the terminal residue of both N-linked and O-linkedoligosaccharides. Variables such as protein structure and cell typeinfluence the number and nature of the carbohydrate units within thechains at different glycosylation sites. Glycosylation isomers are alsocommon at the same site within a given cell type.

[0042] For example, several types of human interferon are glycosylated.Natural human interferon-α2 is O-glycosylated at threonine 106, andN-glycosylation occurs at asparagine 72 in interferon-α14 (Adolf et al.,J. Biochem 276:511 (1991); Nyman T A et al., J. Biochem 329:295 (1998)).The oligosaccharides at asparagine 80 in natural interferon-β1α may playan important factor in the solubility and stability of the protein, butmay not be essential for its biological activity. This permits theproduction of an unglycosylated analog (interferon-β1b) engineered withsequence modifications to enhance stability (Hosoi et al., J. InterferonRes. 8:375 (1988; Karpusas et al., Cell Mol Life Sci 54:1203 (1998);Knight, J. Interferon Res. 2:421 (1982); Runkel et al., Pharm Res 15:641(1998); Lin, Dev. Biol. Stand. 96:97 (1998))1. Interferon-y contains twoN-linked oligosaccharide chains at positions 25 and 97, both importantfor the efficient formation of the bioactive recombinant protein, andhaving an influence on the pharmacokinetic properties of the protein(Sareneva et al., Eur. J. Biochem 242:191 (1996); Sareneva et al.Biochem J. 303:831 (1994); Sareneva et al., J. Interferon Res. 13:267(1993)). Mixed O-linked and N-linked glycosylation also occurs, forexample in human erythropoietin, N-linked glycosylation occurs atasparagine residues located at positions 24, 38 and 83 while O-linkedglycosylation occurs at a serine residue located at position 126 (Lai etal., J. Biol. Chem. 261:3116 (1986); Broudy et al., Arch. Biochem.Biophys. 265:329 (1988)).

[0043] Therapeutic proteins corresponding to a Therapeutic proteinportion of an albumin fusion protein of the invention, as well asanalogs and variants thereof, may be modified so that glycosylation atone or more sites is altered as a result of manipulation(s) of theirnucleic acid sequence, by the host cell in which they are expressed, ordue to other conditions of their expression. For example, glycosylationisomers may be produced by abolishing or introducing glycosylationsites, e.g., by substitution or deletion of amino acid residues, such assubstitution of glutamine for asparagine, or unglycosylated recombinantproteins may be produced by expressing the proteins in host cells thatwill not glycosylate them, e.g. in E. coli or glycosylation-deficientyeast. These approaches are described in more detail below and are knownin the art.

[0044] Therapeutic proteins corresponding to a Therapeutic proteinportion of an albumin fusion protein of the invention include, but arenot limited to, plasma proteins. More specifically, such Therapeuticproteins include, but are not limited to, immunoglobulins, serumcholinesterase, alpha-1 antitrypsin, aprotinin, coagulation factors inboth pre and active forms including but not limited to, von Willebrandfactor, fibrinogen, factor II, factor VII, factor VIIA activated factor,factor VIII, factor IX, factor X, factor XIII, c1 inactivator,antithrombin III, thrombin, prothrombin, apo-lipoprotein, c-reactiveprotein, and protein C. Therapeutic proteins corresponding to aTherapeutic protein portion of an albumin fusion protein of theinvention further include, but are not limited to, human growth hormone(hGH), α-interferon, erythropoietin (EPO), granulocyte-colonystimulating factor (GCSF), granulocyte-macrophage colony-stimulatingfactor (GMCSF), insulin, single chain antibodies, autocrine motilityfactor, scatter factor, laminin, hirudin, applaggin, monocytechemotactic protein (MCP/MCAF), macrophage colony-stimulating factor(M-CSF), osteopontin, platelet factor 4, tenascin, vitronectin, inaddition to those described in Table 1. These proteins and nucleic acidsequences encoding these proteins are well known and available in publicdatabases such as Chemical Abstracts Services Databases (e.g., the CASRegistry), GenBank, and GenSeq as shown in Table 1.

[0045] Additional Therapeutic proteins corresponding to a Therapeuticprotein portion of an albumin fusion protein of the invention include,but are not limited to, one or more of the Therapeutic proteins orpeptides disclosed in the “Therapeutic Protein X” column of Table 1, orfragment or variable thereof.

[0046] Table 1 provides a non-exhaustive list of Therapeutic proteinsthat correspond to a Therapeutic protein portion of an albumin fusionprotein of the invention. The “Therapeutic Protein X” column disclosesTherapeutic protein molecules followed by parentheses containingscientific and brand names that comprise, or alternatively consist of,that Therapeutic protein molecule or a fragment or variant thereof.“therapeutic protein X” as used herein may refer either to an individualTherapeutic protein molecule (as defined by the amino acid sequenceobtainable from the CAS and Genbank accession numbers), or to the entiregroup of Therapeutic proteins associated with a given Therapeuticprotein molecule disclosed in this column. The “Exemplary Identifier”column provides Chemical Abstracts Services (CAS) Registry Numbers(published by the American Chemical Society) and/or Genbank AccessionNumbers ((e.g., Locus ID, NP_XXXXX (Reference Sequence Protein), andXP_XXXXX (Model Protein) identifiers available through the nationalCenter for Biotechnology Information (NCBI) webpage atwww.ncbi.nlm.nih.gov) that correspond to entries in the CAS Registry orGenbank database which contain an amino acid sequence of the TherapeuticProtein Molecule or of a fragment or variant of the Therapeutic ProteinMolecule. The summary pages associated with each of these CAS andGenbank Accession Numbers are each incorporated by reference in theirentireties, particularly with respect to the amino acid sequencesdescribed therein. The “PCT/Patent Reference” column provides U.S.patent numbers, or PCT International Publication Numbers correspondingto patents and/or published patent applications that describe theTherapeutic protein molecule. Each of the patents and/or publishedpatent applications cited in the “PCT/Patent Reference” column areherein incorporated by reference in their entireties. In particular, theamino acid sequences of the specified polypeptide set forth in thesequence listing of each cited “PCT/Patent Reference”, the variants ofthese amino acid sequences (mutations, fragments, etc.) set forth, forexample, in the detailed description of each cited “PCT/PatentReference”, the therapeutic indications set forth, for example, in thedetailed description of each cited “PCT/Patent Reference”, and theactivity asssays for the specified polypeptide set forth in the detaileddescription, and more particularly, the examples of each cited“PCT/Patent Reference” are incorporated herein by reference. The“Biological activity” column describes Biological activities associatedwith the Therapeutic protein molecule. The “Exemplary Activity Assay”column provides references that describe assays which may be used totest the therapeutic and/or biological activity of a Therapeutic proteinor an albumin fusion protein of the invention comprising a Therapeuticprotein X portion. Each of the references cited in the “ExemplaryActivity Assay” column are herein incorporated by reference in theirentireties, particularly with respect to the description of therespective activity assay described in the reference (see Methodssection, for example) for assaying the corresponding biological activityset forth in the “Biological Activity” column of Table 1. The “PreferredIndication Y” column describes disease, disorders, and/or conditionsthat may be treated, prevented, diagnosed, or ameliorated by Therapeuticprotein X or an albumin fusion protein of the invention comprising aTherapeutic protein X portion. Exemplary PCT/Patent Therapeutic ProteinX Identifier Reference Biological Activity Exemplary Activity AssayPreferred Indication Y Alpha-1-antitrypsin CAS-9041-92-3 WO8600337Alpha-1-antitrypsin is an an Enzyme inhibition asay: Gaillard Emphysema;Infant (Alpha-1 proteinase; LocusID: 5265 EP103409-A enzyme inhibitorthat belongs to MC, Kilroe-Smith TA. 1987 Respiratory DistressAlpha-1-trypsin LocusID: 5299 EP155188 the family of serpin serineDetermination of functional activity Syndrome; Pulmonary inhibitor;Prolastin; NP_000286 US5399684 protease inhibitors. The molecule ofalpha 1-protease inhibitor and Fibrosis; Respiratory API; API Inhale)NP_006211 US5736379 inhibits the activity of trypsin alpha2-macroglobulin in human Syncytial Virus XP_007481 US6025161 andelastase. plasma using elastase. J Clin Chem Infections; Asthma; CysticXP_012372 US4839283 Clin Biochem. 25(3): 167-72. Fibrosis; GenitourinaryUS4876197-A Burnouf T, Constans J, Clerc A, Disorders; HIV InfectionsDescamps J, Martinache L, Treatment; Inflammatory Goudemand M. 1987Biochemical Bowel Disorders; Skin and biological properties of an alphaDisorders; Viral Hepatitis; 1-antitrypsin concentrate. Vox Alpha-1Antitrypsin Sang; 52(4): 291-7 Deficiency; Adult Respiratory DistressSyndrome Antihemophilic factor LocusID: 7450 WO8606096 The glycoproteinencoded by this gene H1 Macroscopic platelet agglutination assay (WrightHemophilia; Hemophilia A; (Von Willebrand factor; NP_000543 EP197592functions as both an R. Ann Clin Lab Sci von Willebrand disease VonWillebrand factor XP_006947 WO9316709-A antihemophilic factor carrierand a 1990 20(1): 73). complex; HELIXATE- US5849536 platelet-vessel wallmediator in Collagen binding assay for von FS; HEMOFIL M; US6008193 theblood coagulation system. It Willebrand factor (Favaloro EJ.KOATE-DVI/HP; US5849702 is crucial to the hemostasis Thromb Haemost 200083(1): 127) ALPHANATE; US5238919 process. Mutations in this geneMONARC-M; or deficiencies in this protein HUMATE-P) result in vonWillebrand's disease. Antithrombin III CAS-155319-91-8 WO9100291 Serpinserine protease that Thrombin activity assay (Verheul et Sepsis;Thrombosis; Unstable (ATIII; Antithrombin- CAS-52014-67-2 EP568833inhibits thrombin and other al., Blood 96: 4216-4221, 2000) AnginaPectoris; Coagulation heparin conjugate; LocusID: 462 GB2116183 proteinsinvolved in blood disorders; Respiratory Distress ATH; aaATIII NP_000479coagulation Syndrome; Control of blood (Antithrombin III- XP_001452clotting during coronary artery modified); Atnativ; bypass surgery;Cancer Anthrobin; Atenativ; (aaATIII) Athimbin; Kybernin; Thrombhibin)Apo-lipoprotein (Apo CAS-150287-52-8 WO8803166-A ApoA1 promotescholesterol Cholesterol efflux from human Atherosclerosis; Coronary E;Apo A4; Apo A1; LocusID: 335 WO9307165-A efflux from tissues to theliver for fibroblasts can be directly measured in response restenosis;Apo B) LocusID: 338 US5408038-A excretion. ApoA1 is the major to lipidreconstituted Hypercholesterolemia; LocusID: 348 WO9107505-A proteincomponent of high ApoA1 (J Biol Chem 1996 Oct Hyperlipidemia; Kaposi'sNP_000030 WO9315198-A density lipoprotein (HDL) in the 11; 271(41):25145-51). The capacity Sarcoma NP_000375 US5472858-A plasma. ApoA1 is acofactor for of ApoB to participate in LDL NP_000032 US5364793-Alecithin cholesterolacyltransferase clearance can be evaluated byXP_006435 WO8702062-A (LCAT), which is responsible for measurements ofApoB binding to XP_002288 WO9307165-A the formation of most plasmahepatic lipase (J Biol Chem, Vol. XP_008844 WO9856938-A1 cholesterylesters. Defects in the 273, Issue 32, 20456-20462) and US4943527-A ApoA1gene are associated with lipoprotein lipase (J Biol Chem. HDL deficiencyand Tangier 1995 Apr 7; 270(14): 8081-6.). ApoE disease. ApoB is themain binding to its receptor can be apolipoprotein of chylomicronsmeasured directly for example as and low density lipoproteinsillustrated for the liver ApoE (LDL). ApoB binds triglycerides receptor(J Biol Chem 1986 Mar and clears LDL from circulation. 25; 261(9):4256-67). ApoE is a component of lipoproteins and a ligand for lowdensity lipoprotein receptor. ApoE binds to a specific receptor on livercells and peripheral cells. ApoE is essential for the normal catabolismof triglyceride-rich lipoprotein constituents. Defects in ApoE result infamilial dysbetalipoproteinemia, or type III hyperlipoproteinemia (HLPIII), in which increased plasma cholesterol and triglycerides are theconsequence of impaired clearance of chylomicron and VLDL remnants.Applaggin CAS-129037-76-9 WO9409036-A Applaggin (Agkistrodon Applagginactivity may be assayed Thrombosis; Stroke; Ischemic Heart (Agkistrodonpiscivorus Genbank: A33990 WO9008772-A piscivorus piscivorus platelet invitro by measuring inhibition of Disorders piscivorus (North WO9210575-Aaggregation inhibitor) is a platelet serotonin release induced byAmerican water JP05255395-A 17,700-Da polypeptide dimer ADP,gamma-thrombin, and moccasin snake); which a potent inhibitor ofcollagen. (Proc Natl Acad Sci USA disulfide-linked Arg- plateletactivation. Applaggin 1989 Oct; 86(20): 8050-4). Gly-Asp-containingblocks platelet aggregation dimeric polypeptide) induced by ADP,collagen, thrombin, or arachidonic acid. This inhibition is found tocorrelate with inhibition of thromboxane A2 generation and of densegranule release of serotonin. Autocrine motility LocusID: 2821WO8707617-A Glucose phosphate isomerase Cell motility assay on mouseCT-26 Hemolytic anemia; factor (AMF; NP_000166 WO9909049-A1(neuroleukin); neurotrophic factor cells (Sun et al., Proc. Natl. Acad.glucosephosphate isomerase phosphoglucose XP_012854 and lymphokine;bladder cancer Sci. USA 96: 5412-5417, 1999; Lin deficiency; Hydropsfetalis isomerase; glucose diagnostic et al., Mol. Cell. Endocrinol. 84:47-54, phosphate isomerase; 1992) neuroleukin) C1 Inactivator (C1CAS-80295-38-1 US5622930-A Activation of complement is an C1 inhibitorfunction can be Angioedema; Pancreatic esterase inhibitor; LocusID: 710WO9106650-A essential part of the mechanism assessed by measurement ofDisorders; Reperfusion Injury; Berinert; Complement NP_000053 ofpathogenesis of a large number of inhibition of complement C1 TransplantRejection; Vascular C1 inactivator; C1 XP_006339 human diseases.C1-esterase cleavage (J Immunol 1994 Mar Disorders Inattivatore Umano)inhibitor (C1-INH) concentrate 15; 152(6): 3199-209) prepared from humanplasma is being successfully used for the treatment of hereditaryangioneurotic edema. Recently, C1-INH has been found to be consumed insevere inflammation and has been shown to exert beneficial effects inseveral inflammatory conditions such as human sepsis, post-operativemyocardial dysfunction due to reperfusion injury, severe capillaryleakage syndrome after bone marrow transplantation, reperfusion injuryafter lung transplantation, burn, and cytotoxicity caused by IL-2therapy in cancer, is a major inhibitor of two pro- inflammatory plasmacascade systems, the classical pathway of complement and the contactactivation system. During the activation of classical pathway, C1-INHinteracts with the activated C1 and inhibits it. Interaction of C1-INHwith activated C1 complex leads to the dissociation of the C1q subunitand formation Coagulant Complex Control of spontaneous (Anti-Inhibitorbleeding in hemophilia A and Coagulant Complex; B; prevention ofbleeding in FEIBA VH; patients on Factor VIII AUTOPLEX T) inhibitorsC-reactive protein LocusID: 1401 WO9221364-A Acute-phase serum proteinthat Platelet activation (Simpson RM, NP_000558 WO9505394-A bindsmicrobial polysaccharides and Immunology 1982 47(1): 193) XP_001859ligands on damaged cells, Platelet aggregation (Cheryk LA. Vet Immunolactivates the classical Immunopathol 1996 complement pathway 52: 27).Increased production of IL-1 alpha, IL-1 beta, and TNF-alpha inmacrophages (Galve-de Rochemonteix B. J Leukoc Biol 1993 53: 439) EPO(Erythropoietin; CAS-113427-24-0 WO9902710-A1 Hormone that senses andCell proliferation assay using a Anemia; Bleeding Disorders Epoetinalfa; Epoetin CAS-122312-54-3 WO8502610-A regulates the level of oxygenin erythroleukemic cell line TF-1. beta; Gene-activated LocusID: 2056WO8603520-A the blood by modulating the (Kitamura et al. 1989 J.Cell.erythropoietin; NP_000790 WO9206116- number of circulating Physiol. 140:323) Darbepoetin-alpha; XP_011627 AWO9206116-A erythrocytes NESP;Epogen; Procrit; US5985607-A Eprex; Erypo; Espo; EP232034 Epoimmun;EPOGIN; NEORECORMON; HEMOLINK; Dynepo; ARANESP) Factor IX (CoagulationCAS-181054-95-5 WO8505125-A Coagulation factor IX is a Factor IXclotting activity: Valder Hemophilia B; bleeding; factor IX (human);LocusID: 2158 WO8505376-A vitamin K-dependent factor that circulates R.et al., 2001 “Posttranslational Factor IX deficiency; Factor IX Complex;NP_000124 WO9747737-A1 in the blood as an modifications of recombinantChristmas disease; bleeding Christmas factor; XP_010270 EP162782-Ainactive zymogen. Factor IX is myotube-synthesized human factor episodesin patients with plasma thromboplastin WO8400560-A converted to anactive form by IX” Blood 97: 130-138. factor VIII inhibitor or FactorVII component (PTC), US4994371-A factor XIa, which excises thedeficiency prothrombin complex activation peptide and thus concentrate(PCC); generates a heavy chain and a Nonacog alpha; light chain heldtogether by one MONONINE; or more disulfide bonds. In the ALPHANINE-SD;blood coagulation cascade, BEBULIN; PROPLEX- activated factor IXactivates factor T; KONYNE; X to its active form through PROFILNINE SD;interactions with Ca+2 ions, BeneFIX; IMMUNINE membrane phospholipids,and VH) factor VIII. Alterations of this gene, including pointmutations, insertions and deletions, cause factor IX deficiency, whichis a recessive X-linked disorder, also called hemophilia B or Christmasdisease. Factor VII (Coagulation CAS-102786-61-8 WO8400560-A Coagulationfactor VII is a Coagulation Assay using Bleeding Disorders; CoronaryFactor VII; Active-site LocusID: 2155 WO9323074-A vitamin K-dependentfactor Prothrombin Clotting Time Restenosis; Hemophilia A and B; Liverinactivated factor VII NP_000122 US5997864-A essential for hemostasis.This (Belaaouaj AA et al., J. Biol. Chem. Disorders; (DEGR-VIIa/FFR-NP_062562 US5580560-A factor circulates in the blood in a 275: 27123-8,2000; Diaz-Collier JA Thrombosis; Vascular VIIa); Eptacog alfa;XP_007179 US4994371-A zymogen form, and is converted et al., ThrombHaemost 71: 339-46, Restenosis; Surgery-related Coagulation FactorXP_007180 EP200421-A to an active form by either factor 1994).hemorrhagic episodes VIIa; Novoseven; WO9427631-A IXa, factor Xa, factorXIIa, or NiaStase; Novostase; WO9309804-A thrombin by minor proteolysis.MONOCLATE-P) Upon activation of the factor VII, a heavy chain containinga catalytic domain and a light chain containing 2 EGF-like domains aregenerated, and two chains are held together by a disulfide bond. In thepresence of factor III and calcium ions, the activated factor thenfurther activates the coagulation cascade by converting factor IX tofactor IXa and/or factor X to factor Xa. Defects in this gene can causecoagulopathy. Factor VIII (Factor VIII; CAS-139076-62-3 WO9621035-A2This gene encodes coagulation Development of a simple Hemophilia A;Hemophilia; Octocog alfa, LocusID: 2157 WO9703195-A1 factor VIII, whichparticipates in chromogenic factor VIII assay for Surgery-relatedhemorrhagic Moroctocog alfa; NP_000123 WO9800542-A2 the intrinsicpathway of blood clinical use. episodes Recombinant XP_013124 EP160457-Acoagulation; factor VIII is a Wagenvoord RJ, Hendrix HH, Antihemophilicfactor; EP160457-A cofactor for factor IXa which, in Hemker HC.Haemostasis Nordiate; ReFacto; WO9959622-A1 the presence of Ca+2 and1989; 19(4): 196-204. Kogenate; Kogenate EP253455-A phospholipids,converts factor X SF; Helixate; to the activated form Xa. ThisRecombinate) gene produces two alternatively spliced transcripts.Transcript variant I encodes a large glycoprotein, isoform a, whichcirculates in plasma and associates with von Willebrand factor in anoncovalent complex. This protein undergoes multiple cleavage events.Transcript variant 2 encodes a putative small protein, isoform b, whichconsists primarily of the phospholipid binding domain of factor VIIIc.This binding domain is essential for coagulant activity. Defects in thisgene results in hemophilia A, a common recessive X-linked coagulationdisorder. Factor X LocusID: 2159 WO9204378-A Encodes the vitaminK-dependent FACTOR X ACTIVITY ASSAY. Van Wijk EM Factor X deficiency;Stuart- NP_000495 WO9309804-A coagulation factor X precursor of et al. Arapid manual Prower factor deficiency; XP_007182 US4994371-A the bloodcoagulation cascade. chromogenic factor X assay. Thromb hemorrhage;menorrhagia; This factor precursor is converted Res 22, 681-686 (1981).hematuria; hemarthrosis to a mature two-chain form by the excision ofthe tripeptide RKR. Two chains of the factor are held together by 1 ormore disulfide bonds; the light chain contains 2 EGF-like domains, whilethe heavy chain contains the catalytic domain which is structurallyhomologous to those of the other hemostatic serine proteases. The maturefactor is activated by the cleavage of the activation peptide by factorIXa (in the intrinsic pathway), or by factor VIIa (in the extrinsicpathway). The activated factor then converts prothrombin to thrombin inthe presence of factor Va, Ca+2, and phospholipid during blood clotting.Mutations of this gene result in factor X deficiency, a hemorrhagiccondition of variable severity. Factor XIII LocusID: 2162 WO9116931-ACoagulation factor XIII is the last BLOOD COAGULATION ASSAY. Karpati L,Factor XIII deficiency; LocusID: 2163 EP494702-A zymogen to becomeactivated in Penke B, Katona E, bleeding tendency; defective LocusID:2164 US7425887-A the blood coagulation cascade. Balogh I, Vamosi G,Muszbek L. wound healing; habitual LocusID: 2165 WO9102536-A Plasmafactor XIII is a A modified, optimized kinetic abortion NP_000120WO9918200-A heterotetramer composed of 2 A photometric assay for theNP_001985 subunits and 2 B subunits. The determination of bloodcoagulation XP_004467 A subunits have catalytic factor XIII activity inplasma. Clin XP_001350 function, and the B subunits do Chem. 2000 Dec;46(12): 1946-55. not have enzymatic activity and may serve as a plasmacarrier molecules. Platelet factor XIII is comprised only of 2 Asubunits, which are identical to those of plasma origin. Upon activationby the cleavage of the activation peptide by thrombin and in thepresence of calcium ion, the plasma factor XIII dissociates its Bsubunits and yields the same active enzyme, factor XIIIa, as plateletfactor XIII. This enzyme acts as a transglutaminase to catalyze theformation of gamma- glutamyl-epsilon-lysine crosslinking between fibrinmolecules, thus stabilizing the fibrin clot. Factor XIII deficiency isclassified into two categories: type I deficiency, characterized by thelack of both the A and B subunits; and type II deficiency, characterizedby the lack of the A subunit alone Fibrinogen; thrombin; LocusID: 2243US6083902-A Following vascular injury, Fibrinogen assay: Halbmayer WM,Tissue adhesion; thrombosis; bleeding aprotinin (Human LocusID: 2244WO9523868-A1 fibrinogen is cleaved by thrombin Haushofer A, Schon R,Radek J, disorders; wounds; fibrinogen; human LocusID: 2266 WO9416085-Ato form fibrin, which is the most Fischer M. Comparison of a newthrombocytopenia; thrombin; aprotinin; LocusID: 2147 WO9523868-A1abundant component of blood automated kinetically determineddysfibrinogenemia; and calcium chloride; NP_000499 WO9523868-A1 clots.In addition, various fibrinogen assay with the 3 mosthypofibrinogenemia; synthocytes; FAMs; NP_068657 WO9529686-A1 cleavageproducts of fibrinogen used fibrinogen assays (functional,afibrinogenemia; renal BERIPLAST-P) NP_005132 US6083902-A and fibrinregulate cell adhesion derived and nephelometric) in amyloidosis;thrombosis; NP_000500 WO9523868-A1 and spreading, display Austrianlaboratories in several dysprothrombinemia NP_068656 WO9528946-A1vasoconstrictor and chemotactic clinical populations and healthyNP_000497 WO9313208-A activities, and are mitogens for controls.Haemostasis 1995 May- US5502034-A several cell types. Coagulation Jun;25(3): 114-23. Tan V, Doyle CJ, US5476777-A factor II is proteolyticallycleaved Budzynski AZ. Comparison of the US6110721-A to form thrombin inthe first step kinetic fibrinogen assay with the von of the coagulationcascade, which Clauss method and the clot recovery ultimately results inthe method in plasma of patients with stemming of blood loss. F2 alsoconditions affecting fibrinogen plays a role in maintainingcoagulability. Am J Clin Pathol. vascular integrity. 1995 Oct; 104(4):455-62. Lawrie AS, McDonald SJ, Purdy G, Mackie IJ, Machin SJ.Prothrombin time derived fibrinogen determination on Sysmex CA-6000. JClin Pathol. 1998 Jun; 51(6): 462-6. Aprotinin assay: Cardigan RA,Mackie IJ, Gippner-Steppert C, Jochum M, Royston D, Gallimore MJ.Determination of plasma aprotinin levels by functional and immunologicassays. Blood Coagul Fibrinolysis. 2001 Jan; 12(1): 37-42. Thrombinassay: Syed S, R[4]C PD, Kulczycky M, Sheffield WP. Potent antithrombinactivity and delayed clearance from the circulation characterizerecombinant hirudin genetically fused to albumin. Blood. 1997 May 1;89(9): 3243-52. G-CSF (Granulocyte CAS-121181-53-1 WO8604506-AStimulates the proliferation and Proliferation of murine NFS-60 cellsChemoprotection; colony-stimulating CAS-135968-09-1 EP220520-Adifferentiation of the progenitor (Weinstein et al, Proc Natl Acad SciInflammatory disorders; factor; Granulokine; CAS-130120-55-7 WO8604506-Acells for granulocytes USA 1986; 83, pp5010-4) Myelocytic leukemia; KRN8601; Filgrastim; CAS-130120-54-6 WO8701132-A Primary neutropenias(e.g.; Lenograstim; CAS-134088-74-7 US6054294-A Kostmann syndrome) orMeograstim; LocusID: 1440 secondary neutropenia; Nartograstim; NP_000750Prevention of neutropenia; Neupogen; NOPIA; XP_008227 Prevention andtreatment of Gran; GRANOCYTE; neutropenia in HIV-infected Granulokine;patients; Infections associated Neutrogin, Neu-up; with neutropenias;Neutromax) Myelopysplasia; Autoimmune disorders GM-CSF (Granulocyte-CAS-99283-10-0 WO8805786 Regulates hematopoietic cell Colony StimulatingAssay: Testa, Bone Marrow Disorders; Bone marrow macrophage colony-CAS-123774-72-1 WO8600639 differentiation, gene expression, N.G., etal., “Assays for transplant; stimulating factor; CAS-60154-12-3WO8603225 growth hematopoietic growth factors.” Chemoprotection;Hepatitis rhuGM-CSF, BI CAS-137463-76-4 US5391706 Balkwill FR (edt)Cytokines, A C; HIV Infections; Lung 61012; Prokine; LocusID: 1437US5545536 practical Approach, pp 229-44; IRL Cancer; Malignant melanoma;Molgramostim; NP_000749 Press Oxford 1991. Mycobacterium aviumSargramostim; GM- XP_003751 complex; Mycoses; Myeloid CSF/IL 3 fusion;Leukemia; Neonatal Milodistim; infections; Neutropenia; OralLeucotropin; mucositis; Prostate Cancer; PROKINE; Stem CellMobilization; LEUKOMAX; Vaccine Adjuvant; Venous Interberin; Leukine;Stasis Ulcers; Prevention of Leukine Liquid; neutropenia; AcutePixykine) myelogenous leukemia; Hematopoietic progenitor cellmobilization; Non-Hodgkin's lymphoma; Acute lymphoblastic leukemia;Hodgkin's disease; Accelerated myeloid recovery; Xenotransplantrejection Hepatitis IG (Hepatitis Exposure to Hepatitis B B ImmuneGlobulin; (HBsAg) or Hepatitis C; Hepatitis C immune perinatal exposureof infants globulin; HCVIG; with HBV or HCV infected BAYHEP; NABI-HB,mothers; ; sexual or household NABI-CIVACIR) exposure to patient withacute HBV or HCV Hepatocyte growth LocusID: 3082 JP03 130091-A HGFdisrupts desmosomal Adams JC et al Production of scatter factor byAlopecia; Cancer; factor (HGF; Scatter Genbank: CAA34387 JP10070990-Ajunctions between epithelial cells ndk, a strain of epithelialChemoprotection; Cirrhosis; factor, SF; HGF/SF) WO9323541-A and inducesa motile fibroblast- cells, and inhibition of scatter factorHaematological disorders; like phenotype in individual activity bysuramin. Journal of Cell Radioprotection cells. The factor thereforealso Science 98: 385-94 (1991); Bhargava influences the invasive growthof MM et al Purification, tumor cells derived from characterization andmechanism of epithelial cells and may be action of scatter factor fromhuman involved also in processes of placenta. Experientia Suppl. 59:63-75 Wound healing and early (1991); Coffer A et al embryonicdevelopment. For Purification and characterization of some cell typesincluding biologically active scatter factor from keratinocytes andmammary ras-transformed NIH-3T3 epithelial cells HGF is merely aconditioned medium. Biochemical motility factor. It is also an Journal278: 35-41 (1991); Dowrick autocrine modulator that PG et al Scatterfactor affects major influences the motility of the changes in thecytoskeletal cells that produce it. It is a potent organization ofepithelial cells mitogen for hepatocytes and also Cytokine 3: 299-310(1991); a morphogen (see: HGF, Furlong RA et al Comparison of hepatocytegrowth factor). HGF biological and immunochemical binds to heparin andthis may be properties indicates that scatter factor important for itsactivities in and hepatocyte growth factor are vivo. The actions of HGFare indistinguishable. Journal of Cell inhibited by Suramin. HGF hasScience 100: 173-7 (1991); Gherardi been shown to be an E et alPurification of scatter factor, Angiogenesis factor in vivo. It afibroblast-derived basic protein that induces cultured microvascularmodulates epithelial interactions and endothelial cells to accumulatemovement. Proceedings of the and secrete significantly increasedNational Academy of Science (USA) quantities of urokinase, an enzymeassociated with development of an invasive endothelial phenotype duringangiogenesis. HGF Hirudin (Lepirudin; CAS-138068-37-8 WO8504418-AHirudin is a potent anticoagulant which Hirudin activity can be measuredCoronary restenosis; Deep Desirudin; Refludan; CAS-120993-53-5EP200655-A inhibits thrombin. using a platelet aggregation assay VeinThrombosis; Revasc) CAS-8001-27-2 EP503829-A (Blood Coagul Fibrinolysis1996 Disseminated Intravascular Genbank: AAA29195 WO9207874-A Mar; 7(2):259-61). Coagulation; Heparin-induced Genbank: AAA01384 WO9201712-Athrombocytopenia and EP340170-A thrombosis syndrome; EP341215-AMyocardial infarction; WO9207874-A Unstable Angina Pectoris; WO9201712-AAnticoagulant in adults suffering from acute coronary syndrome;Thrombosis; Veinous Thrombosis Human growth CAS-82030-87-3 WO9418227-APlays an important role in growth control; Ba/F3-hGHR proliferationassay, a Acromegaly; Growth failure; hormone (Pegvisamont;CAS-12629-01-5 WO9005185-A binds 2 GHR molecules novel specific bioassayfor serum Growth failure and Somatrem; Somatropin; LocusID: 2688WO9520398-A and induces signal transduction human growth hormone. J Clinendogenous growth hormone TROVERT; LocusID: 2689 EP245138-A throughreceptor dimerization Endocrinol Metab 2000 replacement; Growth hormonePROTROPIN; BIO- NP_000506 WO8605804-A Nov; 85(11): 4274-9 deficiency;Growth failure and TROPIN; NP_072053 WO9004788-A Plasma growth hormone(GH) growth retardation Prader- HUMATROPE; NP_072054 WO9418227-A1immunoassay and tibial bioassay, Willi syndrome in children 2 NUTROPIN;NP_072055 US6013579-A Appl Physiol 2000 Dec; 89(6): 2174-8 years orolder; Growth NUTROPIN AQ; NP_072056 US6194176-A deficiencies;Postmenopausal NUTROPHIN; NP_002050 WO8605804 Growth hormone (hGH)receptor osteoporosis; burns; cachexia; NORDITROPIN; NP_072050 US6110707mediated cell mediated proliferation, cancer cachexia; dwarfism;GENOTROPIN; NP_072051 US4977089 Growth Horm IGF Res 2000 metabolicdisorders; obesity; SAIZEN; SEROSTIM) NP_072052 US5580723 Oct; 10(5):248-55 renal failure; Turner's XP_008250 US5955346 Internationalstandard for growth Syndrome; fibromyalgia; US6013478 hormone, Horm Res1999; 51 Suppl fracture treatment; frailty WO8605804 1: 7-12 WO9004788-AWO9418227-A1 US6110707-A US4977089 US5580723 US5955346 US6013478WO8605804 US6110707 US5580723 US5955346 US6013478 WO8605804 WO9004788-AWO9418227-A1 US6110707-A US4977089 US5580723 US5955346 US6013478 Insulin(Human insulin; CAS-11061-68-0 WO200040613- Insulin is a heterodimericInsulin activity may be assayed in Hyperglycemia; Diabetes Insulinaspart; Insulin CAS-116094-23-6 A1 polypeptide hormone involved in vitrousing a [3-H]-glucose uptake mellitus; Type 1 diabetes and Glargine;Insulin lispro; CAS-133107-64-9 EP37723-A carbohydrate metabolism. Afterassay. (J Biol Chem 1999 Oct 22; type 2 diabetes Lys-B28 Pro-B29;CAS-160337-95-1 EP55942-A removal of the precursor signal 274(43):30864-30873). lyspro; LY 275585; LocusID: 3630 US4431740-A peptide,proinsulin is post- diarginylinsulin; Des- NP_000198 US4430266-Atranslationally cleaved into two B26-B30-insulin-B25- XP_006400US4624926-A chains (peptide A and peptide B) amide; Insulin detemir;US5077204-A that are covalently linked via two LABI; NOVOLIN;US5840542-A disulfide bonds. Binding of this NOVORAPID; US6110707-Amature form of insulin to the HUMULIN; WO9200322-A insulin receptor(INSR) NOVOMIX 30; stimulates glucose uptake. VELOSULIN NOVOLOG; LANTUS;ILETIN; HUMALOG; MACRULIN; EXUBRA; INSUMAN; ORALIN; ORALGEN; HUMAHALE;HUMAHALIN) Interferon alfa CAS-74899-72-2 EP32134 Interferon alphabelongs to the Anti-viral assay: Rubinstein S, Hepatitis C; oncologyuses; (Interferon alfa-2b; CAS-76543-88-9 WO9419373-A type I Interferonfamily of Familletti PC, Pestka S. (1981) cancer; hepatitis; humanrecombinant; Interferon CAS-99210-65-8 WO9201055 functionally relatedcytokines that Convenient assay for interferons. J. papilloma virus;fibromyalgia; alfa-nl; Interferon alfa- LocusID: 3440 US5602232 confer arange of cellular Virol. 37(2): 755-8; Anti- Sjogren's syndrome; hairycell n3; Peginterferon alpha- NP_000596 US6069133 responses includingantiviral, proliferation assay: Gao Y, et al leukemia; chronic 2b;Ribavirin and XP_011801 WO8302461 antiproliferative, antitumor and(1999) Sensitivity of an epstein-barr myelogeonus leukemia; interferonalfa-2b; CAS-9008-11-1 US6069133 immunomodulatory activities.virus-positive tumor line, Daudi, to AIDS-related Kaposi's Interferonalfacon-1; CAS-118390-30-0 US4569908 alpha interferon correlates withsarcoma; chronic hepatitis B; interferon consensus; LocusID: 3439US4758428 expression of a GC-rich viral malignant melanoma; non- YM 643;CIFN, NP_076918 transcript. Mol Cell Biol. Hodgkin's lymphoma;interferon -alpha 19(11): 7305-13. external condylomata consensus;recombinant acuminata; HIV infection; methionyl consensus small celllung cancer; interferon; recombinant hematological malignancies;consensus interferon; herpes simplex virus CGP 35269; RO infections;multiple sclerosis; 253036; RO 258310; viral hemmorhagic fevers; INTRONA; PEG- solid tumors; renal cancer; INTRON; OIF; bone marrow disorders;bone OMNIFERON; PEG- disorders; bladder cancer; OMNIFERON; gastriccancer; Hepatitis D; VELDONA; PEG- multiple myeloma; type I REBETRON;diabetes mellitus; viral ROFERON A; infections; Cutaneous T-cellWELLFERON; lymphoma; Cervical ALFERON N/LDO; dysplasia; Chronic fatigueREBETRON; syndrome; Renal cancer ALTEMOL; VIRAFERONPEG; PEGASYS;VIRAFERON; VIRAFON, AMPLIGEN; INFERGEN; INFAREX; ORAGEN) IVIG(Intravenous Regulates hematopoietic cell Immune deficiencies; ImmuneGlobulin; differentiation, gene expression, agammaglobulinemia;VENOGLOBULIN-S; growth hypogammaglobulinemia; PANGLOBULIN,immunodeficient states and POLYGAM; bacterial infections; KawasakiGAMMAR-P, Syndrome; Hepatitis A; GAMMAGARD S/D; measles varicella;rubella; IVEEGAM; BAYGAN; immunoglobulin deficiency; SANDOGLOBULIN;idiopathic thrombocytopenic GAMIMUNE) purpura; primary humoralimmunodeficiency states; bone marrow transplantation; pediatric HIVinfection; Guillain-Barre syndrome; chronic inflammatory demyelinatingpolyneuropathy; multifocal neuropathy; dermatomyositis; amyotrophiclateral sclerosis; inclusion-body myositis; Lambert-Eaton myasthenicsyndrome; Rasmussen syndrome; West syndrome; intractable childhoodepilepsy; Lennox-Gastaut syndrome; polymyositis; relapsing- remittingmultiple sclerosis; optic neuritis; stiff-man syndrome; paraneuplasticcerebellar degeneration; paraneoplastic encephalomyelitis and sensoryneuropathy; systemic vasculitis; myelopathy associated with human T-celllymphotrophic virus-1 infection. IVIG-CMV Cytomegalovirus disease(Cytomegalovirus immune globulin intravenous (human); CMV_IVIG; CYTOGAM)Laminin LocusID: 3907 WO9506660 Basement membrane protein; cell Neuriteoutgrowth assay, Neurosci LocusID: 3908 US5658789-A adhesion,differentiation, Lett 2001 Mar 30,301(2): 83-6 LocusID: 3909 WO8901493-Amigration, signaling, neurite Cell adhesion assay, CAFCA, LocusID: 3910WO9811217-A2 outgrowth and metastasis Centrifugal Assay forFluorescence- LocusID: 3911 WO9511972-A based Cell Adhesion, Cancer ResLocusID: 3912 WO9111462-A 2001 Jan 1; 61(1): 339-47 LocusID: 3913WO9508628-A2 Cell migration assay, Biochem LocusID: 3914 WO200066732-Biophys Res Commun 2000 Nov LocusID: 3915 A2 30; 278(3): 614-20 LocusID:3918 WO9815179-A1 LocusID: 10319 WO9919348-A1 NP_000417 WO200066730-NP_000218 A2 NP_002281 US7267564-A NP_002282 WO9610646-A1 NP_002283WO200066731- NP_000219 A2 NP_002284 US5658789-A NP_005553 WO200058473-NP_061486 A2 NP_006050 XP_011387 XP_008772 XP_004301 XP_011616 XP_001716XP_002204 XP_002202 XP_002203 XP_011791 MCP/MCAF LocusID: 6347US5714578-A Chemotactic factor for Transendothelial lymphocyte Cancer;Chemoprotection; (Monocyte Chemotactic LocusID: 6355 US7330446-Amonocytes; chemokine involved chemotaxis assay: Carr, M.W., et WoundsProtein; Monocyte LocusID: 6354 US6090795-A in recruiting leukocytesduring al., Proc. Natl. Acad. Sci. USA, chemoattracting NP_002973US7304234-A inflammation; attracts vol. 91, pp. 3652-3656 (Aprilpeptides) NP_005614 US5571713-A macrophages during 1994). NP_006264US5605671-A inflammation and metastasis XP_008415 WO9725427-A1 XP_008412EP906954-A1 XP_012649 WO9912968-A2 WO9509232-A EP488900-A WO9504158-AWO9509232-A M-CSF (Macrophage CAS-148637-05-2 US5171675-A M-CSFstimulates the growth of M-CSF can be assayed in a Colony Cancer;Hypercholesterolemia colony-stimulating LocusID: 1435 US4929700-Amacrophage/granulocyte- formation assay by the development factor;CSF-1; NP_000748 US5573930-A containing colonies in soft agar ofcolonies containing macrophages Cilmostim; Macstim) XP_002150US5672343-A cultures, influences the (Int J Cell Cloning 1984US5681719-A proliferation and differentiation of Nov; 2(6): 356-67).M-CSF is also US5643563-A hematopoietic stem cells into detected inspecific Bioassays with US5861150-A macrophages but mainly the cellslines that depend in their US6117422-A growth survival and growth on thepresence of M-CSF or US6103224-A differentiation of monocytes. In thatrespond to this factor, for US6156300-A combination with another colonyexample, BAC1.2F5; BaF3; US6146851-A stimulating factor, GM-CSF, oneGNFS-60, J774. An alternative and observes the phenomenon of entirelydifferent detection method is synergistic suppression, i. e., the RT-PCRquantitation of cytokines. combination of these two factors leads to apartial suppression of the generation of macrophage- containing cellcolonies. M-CSF is a specific factor in that the proliferation inducingactivity is more or less restricted to the macrophage lineage. M-CSFalso is a potent stimulator of functional activities of monocytes. Innormal human macrophages M-CSF induces antibody-dependent cellularcytotoxicity. In monocytes and macrophages M-CSF induces the synthesisof IL1, G-CSF, IFN, TNF, plasminogen activator, thromboplastin,prostaglandins and thromboxanes. MSF (Migration Genbank: CAC20427WO9931233-A1 Chemotaxis Transendothelial lymphocyte Wound healingstimulating factor) chemotaxis assay: Carr, M.W., et al., Proc. Natl.Acad. Sci. USA, vol. 91, pp. 3652-3656 (April 1994). NCAF (Neutrophilchemoattracting peptides) Osteopontin (OPN, LocusID: 6696 WO9915904-A1Osteopontin (OPN) is a highly Cell Attachment Assay: Senger DR, BoneFractures BNSP, BSPI, ETA- NP_000573 WO200062065- phosphorylatedsialoprotein that Perruzzi CA, Papadopoulos-Sergiou 1,secreted XP_011125A1 is a prominent component of the A, Van de Water L. (1994)phosphoprotein 1, bone WO9222316-A mineralized extracellular matricesAdhesive properties of osteopontin: sialoprotein I, early T- of bonesand teeth. OPN is regulation by a naturally occurring lymphocyteactivation characterized by the presence of a thrombin-cleavage inclose 1) polyaspartic acid sequence and proximity to the GRGDS cell-sites of Ser/Thr phosphorylation binding domain. Mol Biol Cell thatmediate hydroxyapatite 5(5): 565-74 binding, and a highly conserved RGDmotif that mediates cell attachment/signaling. Expression of OPN in avariety of tissues indicates a multiplicity of functions that involveone or more of these conserved motifs. OPN is involved in a range ofbiological activities including developmental processes, wound healing,immunological responses, tumorigenesis, bone resorption, andcalcification. Platelet Factor 4 CAS-37270-94-3 WO9302192-A Plateletfactor 4 (PF-4) is a CXC- Anti-angeogenic assay (PMID: BleedingDisorders; (Endostatin B; Iroplact; LocusID: 5196 WO9504158-A chemokinewith strong anti- 11259363) Colorectal Cancer; Diabetic RG 1001;Replistatin) NP_002610 US5248666-A angiogenic properties. Retinopathy;Glioma; Heparin XP_003505 US5776892-A Neutralization after CardiacCatheterization or Cardiopulmonary Bypass Surgery; Kaposi's Sarcoma;Malignant Melanoma; Renal Cancer Protein C (Drotrecogin CAS-60202-16-6WO9109953-A Protein C is a serine protease Protein C activity may beassayed in Disseminated intravascular alfa; Activated Protein LocusID:5624 WO9112320-A involved in coagulation and vitro using a coagulationassay. (J coagulation; Septic shock; C, CTC 111; Ceprotin; NP_000303US5516650-A fibrinolysis. Biol Chem 2000 Sep 1; 275(35): ThrombosisrhAPC; Zovant) XP_002706 US5358932-A 27123-27128; Thromb Haemost 1994Mar; 71(3): 339-346). Prothrombin (Factor II, LocusID: 2147 WO9313208-ACoagulation factor II is Prothrombin quantitation and Thrombin, F2)NP_000497 US5502034-A proteolytically cleaved to form activation assay.“CA-1 method, a US5476777-A thrombin in the first step of the novelassay for quantification of US6110721-A coagulation cascade, whichnormal prothrombin using a Ca2+- ultimately results in the dependentprothrombin activator, stemming of blood loss. F2 also carinactivase-1.”Thromb Res. 1999 plays a role in maintaining May 15; 94(4): 221-6.“Activation of vascular integrity. human prothrombin by arginine-specific cysteine proteinases (Gingipains R) from Porphyromonasgingivalis.” J Biol Chem. 2001 Mar 16 Rabies IG (Rabies Rabies ImmuneGlobulin; BAYRAB; HYPERRAB; IMOGAM RABIES- HT; IMOGAM) RhoD IG (RhoDPrevention of Immune Globulin; isoimmunization of RhoD IVIG-Rho(D);negative women at time of PAYRHO-D; spontaneous or induced MICRHOGAM;abortion or transfusion in RHOGAM; WinRho pregnancy; Hemolyic disease;SDF) immune thrombocytopenic purpura; HIV infection RSV IVIG(Respiratory Lower respiratory tract syncytial virus IV infections;respiratory immune globulin syncytial infections (human); Hypermune RSV;RESPIGAM) Serum Cholinesterase LocusID: 590 WO9107483-A Also known asBchE activity assay “Differential LocusID: 1110 WO9523158-Abutyrylcholinesterase/pseuocholin inhibition of human serum NP_000046US6001625-A esterase E1(CHE1). Human cholinesterase with fluoride:XP_003134 US5695750-A tissues have two distinct recognition of two newphenotypes.” cholinesterase activities: Nature 191: 496-498, 1961. “Arare acetylcholinesterase and genetically determined variant ofbutyrylcholinesterase. pseudocholinesterase in two GermanAcetylcholinesterase functions in families with high plasma enzyme thetransmission of nerve activity.” Europ. J. Biochem. 99: impulses,whereas the 65-69, 1979. “Genetic analysis of a physiological functionof butyryl- Japanese patient with cholinesterase remains unknown.butyrylcholinesterase deficiency.” An atypical form of Ann. Hum. Genet.61: 491-496, butyrylcholinesterase or the 1997. absence of its activityleads to prolonged apnea following administration of the muscle relaxantsuxamethonium. The widespread expression of CHE1 in earlydifferentiation suggests development-related functions for this protein.Tenascin LocusID: 7143 WO9628550-A1 The tenascins (TN) are a family celladhesion assay. “Cell adhesion LocusID: 7146 WO9608513-A1 ofextracellular matrix proteins. to fibronectin and tenascin: LocusID:7148 US5681931-A The genes are expressed in quantitative measurements ofinitial NP_003276 US5635360-A distinct tissues at different timesbinding and subsequent NP_009047 WO9222319-A during embyronicdevelopment strengthening response.”J Cell Biol. XP_001730 WO9608513-A1and are present in adult tissues. 1989 Oct; 109(4 Pt 1): 1795-805.;XP_004201 US5681931-A TN-R is detected predominantly “Tenascininterferes with fibronectin US5635360-A in the central nervous system ofaction.” Cell. 1988 May US6048704-A early embryos and likely 6; 53(3):383-90. neurite growth in involved in central nervous vitro. “Tenascinis accumulated system development. TN-XA is along developing peripheralnerves overexpressed in many tumors. and allows neurite outgrowth invitro.” Development. 1990 Oct; 110(2): 401-15. Tetanus IG (TetanusTetanus Immune Globulin; TIG; BAYTET) Vitronectin LocusID: 7448US5514582-A Binds to serpin serine protease Individual functions of themolecule are assayed Atherosclerosis; Vascular Restenosis; NP_000629US6140072-A inhibitors such as PAI, mediates separately. The cellCancer; XP_008484 WO9213075-A cell-to-substrate adhesion, adhesionfunction is assayed using a Cardiovascular Disorders; inhibits thecytolytic action of cell adhesion assay (Feinberg and MalignantMelanoma; the terminal complement cascade Vogelstein, 1983; Anal.Biochem. Clotting disorders; in vitro and inhibits inactivation 132pp6-10); PAI binding using a Transplantation. of thrombin byantithrombin, solid phase binding assay (Seiffert & thereby regulatingcoagulation. Loskutoff, 1991; J. Biol. Chem. 266 pp2824-2830)

[0047] In preferred embodiments, the albumin fusion proteins of theinvention are capable of a therapeutic activity and/or biologic activitycorresponding to the therapeutic activity and/or biologic activity ofthe Therapeutic protein corresponding to the Therapeutic protein portionof the albumin fusion protein listed in the corresponding row ofTable 1. (See, e.g., the “Biological Activity” and “Therapeutic ProteinX”columns of Table 1.) In further preferred embodiments, thetherapeutically active protein portions of the albumin fusion proteinsof the invention are fragments or variants of the reference sequencecited in the “Exemplary Identifier” column of Table 1, and are capableof the therapeutic activity and/or biologic activity of thecorresponding Therapeutic protein disclosed in “Biological Activity”column of Table 1.

[0048] Polypeptide and Polynucleotide Fragments and Variants

[0049] Fragments

[0050] The present invention is further directed to fragments of theTherapeutic proteins described in Table 1, albumin proteins, and/oralbumin fusion proteins of the invention.

[0051] Even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the Therapeutic protein, albumin protein, and/or albuminfusion protein, other Therapeutic activities and/or functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind a ligand) may still be retained. For example, the ability ofpolypeptides with N-terminal deletions to induce and/or bind toantibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

[0052] Accordingly, fragments of a Therapeutic protein corresponding toa Therapeutic protein portion of an albumin fusion protein of theinvention, include the full length protein as well as polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the reference polypeptide (e.g., a Therapeutic proteinas disclosed in Table 1). In particular, N-terminal deletions may bedescribed by the general formula m-q, where q is a whole integerrepresenting the total number of amino acid residues in a referencepolypeptide (e.g., a Therapeutic protein referred to in Table 1), and mis defined as any integer ranging from 2 to q-6. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0053] In addition, fragments of serum albumin polypeptidescorresponding to an albumin protein portion of an albumin fusion proteinof the invention, include the full length protein as well aspolypeptides having one or more residues deleted from the amino terminusof the amino acid sequence of the reference polypeptide (i.e., serumalbumin). In particular, N-terminal deletions may be described by thegeneral formula m-585, where 585 is a whole integer representing thetotal number of amino acid residues in serum albumin (SEQ ID NO:18), andm is defined as any integer ranging from 2 to 579. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0054] Moreover, fragments of albumin fusion proteins of the invention,include the full length albumin fusion protein as well as polypeptideshaving one or more residues deleted from the amino terminus of thealbumin fusion protein. In particular, N-terminal deletions may bedescribed by the general formula m-q, where q is a whole integerrepresenting the total number of amino acid residues in the albuminfusion protein, and m is defined as any integer ranging from 2 to q-6.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0055] Also as mentioned above, even if deletion of one or more aminoacids from the N-terminus or C-terminus of a reference polypeptide(e.g., a Therapeutic protein and/or serum albumin protein) results inmodification or loss of one or more biological functions of the protein,other functional activities (e.g., biological activities, ability tomultimerize, ability to bind a ligand) and/or Therapeutic activities maystill be retained. For example the ability of polypeptides withC-terminal deletions to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking the N-terminal and/or C-terminal residues of areference polypeptide retains Therapeutic activity can readily bedetermined by routine methods described herein and/or otherwise known inthe art.

[0056] The present invention further provides polypeptides having one ormore residues deleted from the carboxy terminus of the amino acidsequence of a Therapeutic protein corresponding to a Therapeutic proteinportion of an albumin fusion protein of the invention (e.g., aTherapeutic protein referred to in Table 1). In particular, C-terminaldeletions may be described by the general formula 1-n, where n is anywhole integer ranging from 6 to q-1, and where q is a whole integerrepresenting the total number of amino acid residues in a referencepolypeptide (e.g., a Therapeutic protein referred to in Table 1).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0057] In addition, the present invention provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of an albumin protein corresponding to an albumin proteinportion of an albumin fusion protein of the invention (e.g., serumalbumin). In particular, C-terminal deletions may be described by thegeneral formula 1-n, where n is any whole integer ranging from 6 to 584,where 584 is the whole integer representing the total number of aminoacid residues in serum albumin (SEQ ID NO:18) minus 1. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0058] Moreover, the present invention provides polypeptides having oneor more residues deleted from the carboxy terminus of an albumin fusionprotein of the invention. In particular, C-terminal deletions may bedescribed by the general formula 1-n, where n is any whole integerranging from 6 to q-1, and where q is a whole integer representing thetotal number of amino acid residues in an albumin fusion protein of theinvention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0059] In addition, any of the above described N- or C-terminaldeletions can be combined to produce a N- and C-terminal deletedreference polypeptide. The invention also provides polypeptides havingone or more amino acids deleted from both the amino and the carboxyltermini, which may be described generally as having residues m-n of areference polypeptide (e.g., a Therapeutic protein referred to in Table1, or serum albumin (e.g., SEQ ID NO:18), or an albumin fusion proteinof the invention) where n and m are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0060] The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto a reference polypeptide sequence (e.g., a Therapeutic protein, serumalbumin protein or an albumin fusion protein of the invention) set forthherein, or fragments thereof. In preferred embodiments, the applicationis directed to proteins comprising polypeptides at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to reference polypeptides having theamino acid sequence of N- and C-terminal deletions as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0061] Preferred polypeptide fragments of the invention are fragmentscomprising, or alternatively, consisting of, an amino acid sequence thatdisplays a Therapeutic activity and/or functional activity (e.g.biological activity) of the polypeptide sequence of the Therapeuticprotein or serum albumin protein of which the amino acid sequence is afragment. Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity.

[0062] Variants

[0063] “Variant” refers to a polynucleotide or nucleic acid differingfrom a reference nucleic acid or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the reference nucleic acid orpolypeptide.

[0064] As used herein, “variant”, refers to a Therapeutic proteinportion of an albumin fusion protein of the invention, albumin portionof an albumin fusion protein of the invention, or albumin fusion proteindiffering in sequence from a Therapeutic protein (e.g. see “therapeutic”column of Table 1), albumin protein, and/or albumin fusion protein ofthe invention, respectively, but retaining at least one functionaland/or therapeutic property thereof (e.g., a therapeutic activity and/orbiological activity as disclosed in the “Biological Activity” column ofTable 1) as described elsewhere herein or otherwise known in the art.Generally, variants are overall very similar, and, in many regions,identical to the amino acid sequence of the Therapeutic proteincorresponding to a Therapeutic protein portion of an albumin fusionprotein of the invention, albumin protein corresponding to an albuminprotein portion of an albumin fusion protein of the invention, and/oralbumin fusion protein of the invention. Nucleic acids encoding thesevariants are also encompassed by the invention.

[0065] The present invention is also directed to proteins whichcomprise, or alternatively consist of, an amino acid sequence which isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to,for example, the amino acid sequence of a Therapeutic proteincorresponding to a Therapeutic protein portion of an albumin fusionprotein of the invention (e.g., an amino acid sequence disclosed in the“Exemplary Identifier” column of Table 1, or fragments or variantsthereof), albumin proteins (e.g., SEQ ID NO:18 or fragments or variantsthereof) corresponding to an albumin protein portion of an albuminfusion protein of the invention, and/or albumin fusion proteins of theinvention. Fragments of these polypeptides are also provided (e.g.,those fragments described herein). Further polypeptides encompassed bythe invention are polypeptides encoded by polynucleotides whichhybridize to the complement of a nucleic acid molecule encoding an aminoacid sequence of the invention under stringent hybridization conditions(e.g., hybridization to filter bound DNA in 6×Sodium chloride/Sodiumcitrate (SSC) at about 45 degrees Celsius, followed by one or morewashes in 0.2×SSC, 0.1% SDS at about 50-65 degrees Celsius), underhighly stringent conditions (e.g., hybridization to filter bound DNA in6×sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius,followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68 degreesCelsius), or under other stringent hybridization conditions which areknown to those of skill in the art (see, for example, Ausubel, F. M. etal., eds., 1989 Current protocol in Molecular Biology, Green publishingassociates, Inc., and John Wiley & Sons Inc., New York, at pages6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides arealso encompassed by the invention.

[0066] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino- or carboxy-terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0067] As a practical matter, whether any particular polypeptide is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence of an albumin fusion protein of theinvention or a fragment thereof (such as the Therapeutic protein portionof the albumin fusion protein or the albumin portion of the albuminfusion protein), can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is expressed as percent identity. Preferredparameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,Gap Size Penalty=0.05, Window Size=500 or the length of the subjectamino acid sequence, whichever is shorter.

[0068] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0069] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0070] The variant will usually have at least 75% (preferably at leastabout 80%, 90%, 95% or 99%) sequence identity with a length of normal HAor Therapeutic protein which is the same length as the variant. Homologyor identity at the nucleotide or amino acid sequence level is determinedby BLAST (Basic Local Alignment Search Tool) analysis using thealgorithm employed by the programs blastp, blastn, blastx, tblastn andtblastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990)and Altschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated byreference) which are tailored for sequence similarity searching.

[0071] The approach used by the BLAST program is to first considersimilar segments between a query sequence and a database sequence, thento evaluate the statistical significance of all matches that areidentified and finally to summarize only those matches which satisfy apreselected threshold of significance. For a discussion of basic issuesin similarity searching of sequence databases, see Altschul et al.,(Nature Genetics 6: 119-129 (1994)) which is fully incorporated byreference. The search parameters for histogram, descriptions,alignments, expect (i.e., the statistical significance threshold forreporting matches against database sequences), cutoff, matrix and filterare at the default settings. The default scoring matrix used by blastp,blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al.,Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated byreference). For blastn, the scoring matrix is set by the ratios of M(i.e., the reward score for a pair of matching residues) to N (i.e., thepenalty score for mismatching residues), wherein the default values forM and N are 5 and -4, respectively. Four blastn parameters may beadjusted as follows: Q=10 (gap creation penalty); R=10 (gap extensionpenalty); wink=1 (generates word hits at every winkth position along thequery); and gapw=16 (sets the window width within which gappedalignments are generated). The equivalent Blastp parameter settings wereQ=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,available in the GCG package version 10.0, uses DNA parameters GAP=50(gap creation penalty) and LEN=3 (gap extension penalty) and theequivalent settings in protein comparisons are GAP=8 and LEN=2.

[0072] The polynucleotide variants of the invention may containalterations in the coding regions, non-coding regions, or both.Especially preferred are polynucleotide variants containing alterationswhich produce silent substitutions, additions, or deletions, but do notalter the properties or activities of the encoded polypeptide.Nucleotide variants produced by silent substitutions due to thedegeneracy of the genetic code are preferred. Moreover, polypeptidevariants in which less than 50, less than 40, less than 30, less than20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a bacterial host, such as, yeast or E.coli).

[0073] In a preferred embodiment, a polynucleotide encoding an albuminportion of an albumin fusion protein of the invention is optimized forexpression in yeast or mammalian cells. In further preferred embodiment,a polynucleotide encoding a Therapeutic protein portion of an albuminfusion protein of the invention is optimized for expression in yeast ormammalian cells. In a still further preferred embodiment, apolynucleotide encoding an albumin fusion protein of the invention isoptimized for expression in yeast or mammalian cells.

[0074] In an alternative embodiment, a codon optimized polynucleotideencoding a Therapeutic protein portion of an albumin fusion protein ofthe invention does not hybridize to the wild type polynucleotideencoding the Therapeutic protein under stringent hybridizationconditions as described herein. In a further embodiment, a codonoptimized polynucleotide encoding an albumin portion of an albuminfusion protein of the invention does not hybridize to the wild typepolynucleotide encoding the albumin protein under stringenthybridization conditions as described herein. In another embodiment, acodon optimized polynucleotide encoding an albumin fusion protein of theinvention does not hybridize to the wild type polynucleotide encodingthe Therapeutic protein portin or the albumin protein portion understringent hybridization conditions as described herein.

[0075] In an additional embodiment, polynucleotides encoding aTherapeutic protein portion of an albumin fusion protein of theinvention do not comprise, or alternatively consist of the naturallyoccurring sequence of that Therapeutic protein. In a further embodiment,polynucleotides encoding an albumin protein portion of an albumin fusionprotein of the invention do not comprise, or alternatively consist of,the naturally occurring sequence of albumin protein. In an alternativeembodiment, polynucleotides encoding an albumin fusion protein of theinvention do not comprise, or alternatively consist of, the naturallyoccurring sequence of a Therapeutic protein portion or the albuminprotein portion.

[0076] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985)). These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0077] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the polypeptide of the present invention withoutsubstantial loss of biological function. As an example, Ron et al. (J.Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins havingheparin binding activity even after deleting 3, 8, or 27 amino-terminalamino acid residues. Similarly, Interferon gamma exhibited up to tentimes higher activity after deleting 8-10 amino acid residues from thecarboxy terminus of this protein. (Dobeli et al., J. Biotechnology7:199-216 (1988).)

[0078] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem.268:22105-22111 (1993)) conducted extensive mutational analysis of humancytokine IL-1a. They used random mutagenesis to generate over 3,500individual IL-1a mutants that averaged 2.5 amino acid changes pervariant over the entire length of the molecule. Multiple mutations wereexamined at every possible amino acid position. The investigators foundthat “[m]ost of the molecule could be altered with little effect oneither [binding or biological activity].” In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0079] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

[0080] Thus, the invention further includes polypeptide variants whichhave a functional activity (e.g., biological activity and/or therapeuticactivity). In highly preferred embodiments the invention providesvariants of albumin fusion proteins that have a functional activity(e.g., biological activity and/or therapeutic activity, such as thatdisclosed in the “Biological Activity” column in Table 1) thatcorresponds to one or more biological and/or therapeutic activities ofthe Therapeutic protein corresponding to the Therapeutic protein portionof the albumin fusion protein. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

[0081] In preferred embodiments, the variants of the invention haveconservative substitutions. By “conservative substitutions” is intendedswaps within groups such as replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

[0082] Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided, for example, in Bowie et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

[0083] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0084] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. See Cunningham and Wells,Science 244:1081-1085 (1989). The resulting mutant molecules can then betested for biological activity.

[0085] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu: replacement of the amide residues Asn and Gin, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acidsubstitution, variants of the present invention include (i) polypeptidescontaining substitutions of one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) polypeptides containingsubstitutions of one or more of the amino acid residues having asubstituent group, or (iii) polypeptides which have been fused with orchemically conjugated to another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), (iv) polypeptide containing additionalamino acids, such as, for example, an IgG Fc fusion region peptide. Suchvariant polypeptides are deemed to be within the scope of those skilledin the art from the teachings herein.

[0086] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

[0087] In specific embodiments, the polypeptides of the inventioncomprise, or alternatively, consist of, fragments or variants of theamino acid sequence of a Therapeutic protein described herein and/orhuman serum albumin, and/or albumin fusion protein of the inventionwherein the fragments or variants have 1-5,5-10, 5-25, 5-50, 10-50 or50-150, amino acid residue additions, substitutions, and/or deletionswhen compared to the reference amino acid sequence. In preferredembodiments, the amino acid substitutions are conservative. Nucleicacids encoding these polypeptides are also encompassed by the invention.

[0088] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

[0089] Functional Activity

[0090] “A polypeptide having functional activity” refers to apolypeptide capable of displaying one or more known functionalactivities associated with the full-length, pro-protein, and/or matureform of a Therapeutic protein. Such functional activities include, butare not limited to, biological activity, antigenicity [ability to bind(or compete with a polypeptide for binding) to an anti-polypeptideantibody], immunogenicity (ability to generate antibody which binds to aspecific polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide.

[0091] “A polypeptide having biological activity” refers to apolypeptide exhibiting activity similar to, but not necessarilyidentical to, an activity of a Therapeutic protein of the presentinvention, including mature forms, as measured in a particularbiological assay, with or without dose dependency. In the case wheredose dependency does exist, it need not be identical to that of thepolypeptide, but rather substantially similar to the dose-dependence ina given activity as compared to the polypeptide of the present invention(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity, and most preferably, not more than aboutthree-fold less activity relative to the polypeptide of the presentinvention).

[0092] In preferred embodiments, an albumin fusion protein of theinvention has at least one biological and/or therapeutic activityassociated with the Therapeutic protein (or fragment or variant thereof)when it is not fused to albumin.

[0093] The albumin fusion proteins of the invention can be assayed forfunctional activity (e.g., biological activity) using or routinelymodifying assays known in the art, as well as assays described herein.Specifically, albumin fusion proteins may be assayed for functionalactivity (e.g., biological activity or therapeutic activity) using theassay referenced in the “Exemplary Activity Assay” column of Table 1.Additionally, one of skill in the art may routinely assay fragments of aTherapeutic protein corresponding to a Therapeutic protein portion of analbumin fusion protein of the invention, for activity using assaysreferenced in its corresponding row of Table 1. Further, one of skill inthe art may routinely assay fragments of an albumin proteincorresponding to an albumin protein portion of an albumin fusion proteinof the invention, for activity using assays known in the art and/or asdescribed in the Examples section below.

[0094] For example, in one embodiment where one is assaying for theability of an albumin fusion protein of the invention to bind or competewith a Therapeutic protein for binding to an anti-Therapeuticpolypeptide antibody and/or anti-albumin antibody, various immunoassaysknown in the art can be used, including but not limited to, competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0095] In a preferred embodiment where a binding partner (e.g., areceptor or a ligand) of a Therapeutic protein is identified, binding tothat binding partner by an albumin fusion protein containing thatTherapeutic protein as the Therapeutic protein portion of the fusion canbe assayed, e.g., by means well-known in the art, such as, for example,reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky et al.,Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability ofphysiological correlates of an albumin fusion protein of the presentinvention to bind to a substrate(s) of the Therapeutic polypeptidecorresponding to the Therapeutic portion of the albumin fusion proteinof the invention can be routinely assayed using techniques known in theart.

[0096] In an alternative embodiment, where the ability of an albuminfusion protein of the invention to multimerize is being evaluated,association with other components of the multimer can be assayed, e.g.,by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky et al., supra.

[0097] In addition, assays described herein (see Examples and Table 1)and otherwise known in the art may routinely be applied to measure theability of albumin fusion proteins of the present invention andfragments, variants and derivatives thereof to elicit biologicalactivity and/or Therapeutic activity (either in vitro or in vivo)related to either the Therapeutic protein portion and/or albumin portionof the albumin fusion protein of the present invention. Other methodswill be known to the skilled artisan and are within the scope of theinvention.

[0098] Albumin

[0099] As described above, an albumin fusion protein of the inventioncomprises at least a fragment or variant of a Therapeutic protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, preferably by genetic fusion or chemical conjugation.

[0100] The terms, human serum albumin (HSA) and human albumin (HA) areused interchangeably herein. The terms, “albumin and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

[0101] As used herein, “albumin” refers collectively to albumin proteinor amino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments thereof (seeEP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the matureform of human albumin as shown in FIG. 15 and SEQ ID NO:18, or albuminfrom other vertebrates or fragments thereof, or analogs or variants ofthese molecules or fragments thereof.

[0102] In preferred embodiments, the human serum albumin protein used inthe albumin fusion proteins of the invention contains one or both of thefollowing sets of point mutations with reference to SEQ ID NO:18:Leu-407 to Ala, Leu-408 to Val, Val409 to Ala, and Arg-410 to Ala; orArg-410 to A, Lys-413 to Gln, and Lys-414 to Gln (see, e.g.,International Publication No. WO95123857, hereby incorporated in itsentirety by reference herein). In even more preferred embodiments,albumin fusion proteins of the invention that contain one or both ofabove-described sets of point mutations have improvedstability/resistance to yeast Yap3p proteolytic cleavage, allowingincreased production of recombinant albumin fusion proteins expressed inyeast host cells.

[0103] As used herein, a portion of albumin sufficient to prolong thetherapeutic activity or shelf-life of the Therapeutic protein refers toa portion of albumin sufficient in length or structure to stabilize orprolong the therapeutic activity of the protein so that the shelf lifeof the Therapeutic protein portion of the albumin fusion protein isprolonged or extended compared to the shelf-life in the non-fusionstate. The albumin portion of the albumin fusion proteins may comprisethe full length of the HA sequence as described above or as shown inFIG. 15, or may include one or more fragments thereof that are capableof stabilizing or prolonging the therapeutic activity. Such fragmentsmay be of 10 or more amino acids in length or may include about 15, 20,25, 30, 50, or more contiguous amino acids from the HA sequence or mayinclude part or all of specific domains of HA. For instance, one or morefragments of HA spanning the first two immunoglobulin-like domains maybe used.

[0104] The albumin portion of the albumin fusion proteins of theinvention may be a variant of normal HA. The Therapeutic protein portionof the albumin fusion proteins of the invention may also be variants ofthe Therapeutic proteins as described herein. The term “variants”includes insertions, deletions and substitutions, either conservative ornon conservative, where such changes do not substantially alter one ormore of the oncotic, useful ligand-binding and non-immunogenicproperties of albumin, or the active site, or active domain whichconfers the therapeutic activities of the Therapeutic proteins.

[0105] In particular, the albumin fusion proteins of the invention mayinclude naturally occurring polymorphic variants of human albumin andfragments of human albumin, for example those fragments disclosed in EP322 094 (namely HA (Pn), where n is 369 to 419). The albumin may bederived from any vertebrate, especially any mammal, for example human,cow, sheep, or pig. Non-mammalian albumins include, but are not limitedto, hen and salmon. The albumin portion of the albumin fusion proteinmay be from a different animal than the Therapeutic protein portion.

[0106] Generally speaking, an HA fragment or variant will be at least100 amino acids long, preferably at least 150 amino acids long. The HAvariant may consist of or alternatively comprise at least one wholedomain of HA, for example domains 1 (amino acids 1-194 of SEQ ID NO:18),2 (amino acids 195-387 of SEQ ID NO:18), 3 (amino acids 388-585 of SEQID NO:18), 1+2 (1-387 of SEQ ID NO:18), 2+3 (195-585 of SEQ ID NO:18) or1+3 (amino acids 1-194 of SEQ ID NO:18+amino acids 388-585 of SEQ IDNO:18). Each domain is itself made up of two homologous subdomainsnamely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, withflexible inter-subdomain linker regions comprising residues Lys106 toGlul 19, Glu292 to Val315 and Glu492 to Ala511.

[0107] Preferably, the albumin portion of an albumin fusion protein ofthe invention comprises at least one subdomain or domain of HA orconservative modifications thereof. If the fusion is based onsubdomains, some or all of the adjacent linker is preferably used tolink to the Therapeutic protein moiety.

[0108] Albumin Fusion Proteins

[0109] The present invention relates generally to albumin fusionproteins and methods of treating, preventing, or ameliorating diseasesor disorders. As used herein, “albumin fusion protein” refers to aprotein formed by the fusion of at least one molecule of albumin (or afragment or variant thereof) to at least one molecule of a Therapeuticprotein (or fragment or variant thereof). An albumin fusion protein ofthe invention comprises at least a fragment or variant of a Therapeuticprotein and at least a fragment or variant of human serum albumin, whichare associated with one another, preferably by genetic fusion (i.e., thealbumin fusion protein is generated by translation of a nucleic acid inwhich a polynucleotide encoding all or a portion of a Therapeuticprotein is joined in-frame with a polynucleotide encoding all or aportion of albumin) or chemical conjugation to one another. TheTherapeutic protein and albumin protein, once part of the albumin fusionprotein, may be referred to as a “portion”, “region” or “moiety” of thealbumin fusion protein.

[0110] In one embodiment, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a Therapeuticprotein (e.g., as described in Table 1) and a serum albumin protein. Inother embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment of a Therapeutic protein and a serumalbumin protein. In other embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, abiologically active and/or therapeutically active variant of aTherapeutic protein and a serum albumin protein. In preferredembodiments, the serum albumin protein component of the albumin fusionprotein is the mature portion of serum albumin.

[0111] In further embodiments, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a Therapeuticprotein, and a biologically active and/or therapeutically activefragment of serum albumin. In further embodiments, the inventionprovides an albumin fusion protein comprising, or alternativelyconsisting of, a Therapeutic protein and a biologically active and/ortherapeutically active variant of serum albumin. In preferredembodiments, the Therapeutic protein portion of the albumin fusionprotein is the mature portion of the Therapeutic protein.

[0112] In further embodiments, the invention provides an albumin fusionprotein comprising, or alternatively consisting of, a biologicallyactive and/or therapeutically active fragment or variant of aTherapeutic protein and a biologically active and/or therapeuticallyactive fragment or variant of serum albumin. In preferred embodiments,the invention provides an albumin fusion protein comprising, oralternatively consisting of, the mature portion of a Therapeutic proteinand the mature portion of serum albumin.

[0113] Preferably, the albumin fusion protein comprises HA as theN-terminal portion, and a Therapeutic protein as the C-terminal portion.Alternatively, an albumin fusion protein comprising HA as the C-terminalportion, and a Therapeutic protein as the N-terminal portion may also beused.

[0114] In other embodiments, the albumin fusion protein has aTherapeutic protein fused to both the N-terminus and the C-terminus ofalbumin. In a preferred embodiment, the Therapeutic proteins fused atthe N- and C-termini are the same Therapeutic proteins. In a preferredembodiment, the Therapeutic proteins fused at the N- and C-termini aredifferent Therapeutic proteins. In another preferred embodiment, theTherapeutic proteins fused at the N- and C-termini are differentTherapeutic proteins which may be used to treat or prevent the samedisease, disorder, or condition (e.g. as listed in the “PreferredIndication Y” column of Table 1). In another preferred embodiment, theTherapeutic proteins fused at the N- and C-termini are differentTherapeutic proteins which may be used to treat or prevent diseases ordisorders (e.g. as listed in the “Preferred Indication Y” column ofTable 1) which are known in the art to commonly occur in patientssimultaneously.

[0115] In addition to albumin fusion protein in which the albuminportion is fused N-terminal and/or C-terminal of the Therapeutic proteinportion, albumin fusion proteins of the invention may also be producedby inserting the Therapeutic protein or peptide of interest (e.g.,Therapeutic protein X as diclosed in Table 1) into an internal region ofHA. For instance, within the protein sequence of the HA molecule anumber of loops or turns exist between the end and beginning ofa-helices, which are stabilized by disulphide bonds (see FIGS. 9-11).The loops, as determined from the crystal structure of HA (FIG. 13) (PDBidentifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E71 and 1UOR) for the mostpart extend away from the body of the molecule. These loops are usefulfor the insertion, or internal fusion, of therapeutically activepeptides, particularly those requiring a secondary structure to befunctional, or Therapeutic proteins, to essentially generate an albuminmolecule with specific biological activity.

[0116] Loops in human albumin structure into which peptides orpolypeptides may be inserted to generate albumin fusion proteins of theinvention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100,Gln170-Ala176, His247-Glu252, Glu266-Glu277, Glu280-His288,Ala362-Glu368, Lys439-Pro447,Val462-Lys475, Thr478-Pro486, andLys560-Thr566. In more preferred embodiments, peptides or polypeptidesare inserted into the Val54-Asn6l, Gln170-Ala176, and/or Lys560-Thr566loops of mature human albumin (SEQ ID NO:18).

[0117] Peptides to be inserted may be derived from either phage displayor synthetic peptide libraries screened for specific biological activityor from the active portions of a molecule with the desired function.Additionally, random peptide libraries may be generated withinparticular loops or by insertions of randomized peptides into particularloops of the HA molecule and in which all possible combinations of aminoacids are represented.

[0118] Such library(s) could be generated on HA or domain fragments ofHA by one of the following methods:

[0119] (a) randomized mutation of amino acids within one or more peptideloops of HA or HA domain fragments. Either one, more or all the residueswithin a loop could be mutated in this manner (for example see FIG.10a);

[0120] (b) replacement of, or insertion into one or more loops of HA orHA domain fragments (i.e., internal fusion) of a randomized peptide(s)of length X_(n) (where X is an amino acid and n is the number ofresidues (for example see FIG. 10b);

[0121] (c) N-, C- or N- and C-terminal peptide/protein fusions inaddition to (a) and/or (b).

[0122] The HA or HA domain fragment may also be made multifunctional bygrafting the peptides derived from different screens of different loopsagainst different targets into the same HA or HA domain fragment. Inpreferred embodiments, peptides inserted into a loop of human serumalbumin are peptide fragments or peptide variants of the Therapeuticproteins disclosed in Table 1. More particulary, the inventionencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acids inlength inserted into a loop of human serum albumin. The invention alsoencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acidsfused to the N-terminus of human serum albumin. The invention alsoencompasses albumin fusion proteins which comprise peptide fragments orpeptide variants at least 7 at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 35, or at least 40 amino acidsfused to the C-terminus of human serum albumin.

[0123] Generally, the albumin fusion proteins of the invention may haveone HA-derived region and one Therapeutic protein-derived region.Multiple regions of each protein, however, may be used to make analbumin fusion protein of the invention. Similarly, more than oneTherapeutic protein may be used to make an albumin fusion protein of theinvention. For instance, a Therapeutic protein may be fused to both theN- and C-terminal ends of the HA. In such a configuration, theTherapeutic protein portions may be the same or different Therapeuticprotein molecules. The structure of bifunctional albumin fusion proteinsmay be represented as: X-HA-Y or Y-HA-X.

[0124] For example, an anti-BLyST™ scFv-HA-IFNα-2b fusion may beprepared to modulate the immune response to IFNα-2b by anti-BLyS™ scFv.An alternative is making a bi (or even multi) functional dose ofHA-fusions e.g. HA-IFNα-2b fusion mixed with HA-anti-BLyS™ scFv fusionor other HA-fusions in various ratio's depending on function, half-lifeetc.

[0125] Bi- or multi-functional albumin fusion proteins may also beprepared to target the Therapeutic protein portion of a fusion to atarget organ or cell type via protein or peptide at the oppositeterminus of HA.

[0126] As an alternative to the fusion of known therapeutic molecules,the peptides could be obtained by screening libraries constructed asfusions to the N-, C- or N- and C-termini of HA, or domain fragment ofHA, of typically 6, 8, 12, 20 or 25 or X_(n) (where X is an amino acid(aa) and n equals the number of residues) randomized amino acids, and inwhich all possible combinations of amino acids were represented. Aparticular advantage of this approach is that the peptides may beselected in situ on the HA molecule and the properties of the peptidewould therefore be as selected for rather than, potentially, modified asmight be the case for a peptide derived by any other method then beingattached to HA.

[0127] Additionally, the albumin fusion proteins of the invention mayinclude a linker peptide between the fused portions to provide greaterphysical separation between the moieties and thus maximize theaccessibility of the Therapeutic protein portion, for instance, forbinding to its cognate receptor. The linker peptide may consist of aminoacids such that it is flexible or more rigid.

[0128] The linker sequence may be cleavable by a protease or chemicallyto yield the growth hormone related moiety. Preferably, the protease isone which is produced naturally by the host, for example the S.cerevisiae protease kex2 or equivalent proteases.

[0129] Therefore, as described above, the albumin fusion proteins of theinvention may have the following formula R1-L-R2; R2-L-R1; orR1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein, peptide orpolypeptide sequence, and not necessarily the same Therapeutic protein,L is a linker and R2 is a serum albumin sequence.

[0130] In preferred embodiments, Albumin fusion proteins of theinvention comprising a Therapeutic protein have extended shelf lifecompared to the shelf life the same Therapeutic protein when not fusedto albumin. Shelf-life typically refers to the time period over whichthe therapeutic activity of a Therapeutic protein in solution or in someother storage formulation, is stable without undue loss of therapeuticactivity. Many of the Therapeutic proteins are highly labile in theirunfused state. As described below, the typical shelf-life of theseTherapeutic proteins is markedly prolonged upon incorporation into thealbumin fusion protein of the invention.

[0131] Albumin fusion proteins of the invention with “prolonged” or“extended” shelf-life exhibit greater therapeutic activity relative to astandard that has been subjected to the same storage and handlingconditions. The standard may be the unfused full-length Therapeuticprotein. When the Therapeutic protein portion of the albumin fusionprotein is an analog, a variant, or is otherwise altered or does notinclude the complete sequence for that protein, the prolongation oftherapeutic activity may alternatively be compared to the unfusedequivalent of that analog, variant, altered peptide or incompletesequence. As an example, an albumin fusion protein of the invention mayretain greater than about 100% of the therapeutic activity, or greaterthan about 105%, 110%, 120%, 130%, 150% or 200% of the therapeuticactivity of a standard when subjected to the same storage and handlingconditions as the standard when compared at a given time point.

[0132] Shelf-life may also be assessed in terms of therapeutic activityremaining after storage, normalized to therapeutic activity when storagebegan. Albumin fusion proteins of the invention with prolonged orextended shelf-life as exhibited by prolonged or extended therapeuticactivity may retain greater than about 50% of the therapeutic activity,about 60%, 70%, 80%, or 90% or more of the therapeutic activity of theequivalent unfused Therapeutic protein when subjected to the sameconditions. For example, as discussed in Example 1, an albumin fusionprotein of the invention comprising hGH fused to the full length HAsequence may retain about 80% or more of its original activity insolution for periods of up to 5 weeks or more under various temperatureconditions.

[0133] Expression of Fusion Proteins

[0134] The albumin fusion proteins of the invention may be produced asrecombinant molecules by secretion from yeast, a microorganism such as abacterium, or a human or animal cell line. Preferably, the polypeptideis secreted from the host cells. We have found that, by fusing the hGHcoding sequence to the HA coding sequence, either to the 5′ end or 3′end, it is possible to secrete the albumin fusion protein from yeastwithout the requirement for a yeast-derived pro sequence. This wassurprising, as other workers have found that a yeast derived prosequence was needed for efficient secretion of hGH in yeast.

[0135] For example, Hiramatsu et al. (Appl Environ Microbiol 56:2125(1990); Appl Environ Microbiol 57:2052 (1991)) found that the N-terminalportion of the pro sequence in the Mucor pusillus rennin pre-pro leaderwas important. Other authors, using the MFα-1 signal, have alwaysincluded the MFα-1 pro sequence when secreting hGH. The pro sequenceswere believed to assist in the folding of the hGH by acting as anintramolecular chaperone. The present invention shows that HA orfragments of HA can perform a similar function.

[0136] Hence, a particular embodiment of the invention comprises a DNAconstruct encoding a signal sequence effective for directing secretionin yeast, particularly a yeast-derived signal sequence (especially onewhich is homologous to the yeast host), and the fused molecule of thefirst aspect of the invention, there being no yeast-derived pro sequencebetween the signal and the mature polypeptide.

[0137] The Saccharomyces cerevisiae invertase signal is a preferredexample of a yeast-derived signal sequence.

[0138] Conjugates of the kind prepared by Poznansky et al., (FEBS Lett.239:18 (1988)), in which separately-prepared polypeptides are joined bychemical cross-linking, are not contemplated.

[0139] The present invention also includes a cell, preferably a yeastcell transformed to express an albumin fusion protein of the invention.In addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. If the polypeptide issecreted, the medium will contain the polypeptide, with the cells, orwithout the cells if they have been filtered or centrifuged away. Manyexpression systems are known and may be used, including bacteria (forexample E. coli and Bacillus subtilis), yeasts (for exampleSaccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris,filamentous fungi (for example Aspergillus), plant cells, animal cellsand insect cells.

[0140] Preferred yeast strains to be used in the production of albuminfusion proteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1,pra1, ubc4] is a derivative of parent strain AH22his⁺ (also known asDB1; see, e.g., Sleep et al. Biotechnology 8:42-46 (1990)). The straincontains a leu2 mutation which allows for auxotropic selection of 2micron-based plasmids that contain the LEU2 gene. D88 also exhibits aderepression of PRB1 in glucose excess. The PRB1 promoter is normallycontrolled by two checkpoints that monitor glucose levels and growthstage. The promoter is activated in wild type yeast upon glucosedepletion and entry into stationary phase. Strain D88 exhibits therepression by glucose but maintains the induction upon entry intostationary phase. The PRA1 gene encodes a yeast vacuolar protease, YscAendoprotease A, that is localized in the ER. The UBC4 gene is in theubiquitination pathway and is involved in targeting short lived andabnormal proteins for ubiquitin dependant degradation. Isolation of thisubc4 mutation was found to increase the copy number of an expressionplasmid in the cell and cause an increased level of expression of adesired protein expressed from the plasmid (see, e.g., InternationalPublication No. WO99/00504, hereby incorporated in its entirety byreference herein).

[0141] DXY1, a derivative of D88, has the following genotype: [leu2-3,leu2-122, can1, pra1, ubc4, ura3:.yap3]. In addition to the mutationsisolated in D88, this strain also has a knockout of the YAP3 protease.This protease causes cleavage of mostly di-basic residues (RR, RK, KR,KK) but can also promote cleavage at single basic residues in proteins.Isolation of this yap3 mutation resulted in higher levels of full lengthHSA production (see, e.g., U.S. Pat. No. 5,965,386, and Kerry-Williamset al., Yeast 14:161-169 (1998), hereby incorporated in their entiretiesby reference herein).

[0142] BXP10 has the following genotype: leu2-3, leu2-122, can1, pra1,ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition tothe mutations isolated in DXY1, this strain also has a knockout of thePMT1 gene and the HSP150 gene. The PMT1 gene is a member of theevolutionarily conserved family of dolichyl-phosphate-D-mannose proteinO-mannosyltransferases (Pmts). The transmembrane topology of Pmt1psuggests that it is an integral membrane protein of the endoplasmicreticulum with a role in O-linked glycosylation. This mutation serves toreduce/eliminate O-linked glycosylation of HSA fusions (see, e.g.,International Publication No. WO00/44772, hereby incorporated in itsentirety by reference herein). Studies revealed that the Hsp150 proteinis inefficiently separated from rHA by ion exchange chromatography. Themutation in the HSP150 gene removes a potential contaminant that hasproven difficult to remove by standard purification techniques. See,e.g., U.S. Pat. No. 5,783,423, hereby incorporated in its entirety byreference herein.

[0143] The desired protein is produced in conventional ways, for examplefrom a coding sequence inserted in the host chromosome or on a freeplasmid. The yeasts are transformed with a coding sequence for thedesired protein in any of the usual ways, for example electroporation.Methods for transformation of yeast by electroporation are disclosed inBecker & Guarente (1990) Methods Enzymol. 194, 182.

[0144] Successfully transformed cells, i.e., cells that contain a DNAconstruct of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anexpression construct can be grown to produce the desired polypeptide.Cells can be harvested and lysed and their DNA content examined for thepresence of the DNA using a method such as that described by Southern(1975) J. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208.Alternatively, the presence of the protein in the supernatant can bedetected using antibodies.

[0145] Useful yeast plasmid vectors include pRS403-406 and pRS413-416and are generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (Ylps) and incorporate the yeast selectable markersHIS3, 7RP 1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (Ycps).

[0146] Preferred vectors for making albumin fusion proteins forexpression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSAwhich are described in detail in Example 2. FIG. 4 shows a map of thepPPC0005 plasmid that can be used as the base vector into whichpolynucleotides encoding Therapeutic proteins may be cloned to formHA-fusions. It contains a PRBI S. cerevisiae promoter (PRBIp), a Fusionleader sequence (FL), DNA encoding HA (rHA) and an ADH1 S. cerevisiaeterminator sequence. The sequence of the fusion leader sequence consistsof the first 19 amino acids of the signal peptide of human serum albumin(SEQ ID NO:29) and the last five amino acids of the mating factor alphaI promoter (SLDKR, see EP-A-387 319 which is hereby incorporated byreference in its entirety.

[0147] The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA weredeposited on Apr. 11, 2001 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209 and given accessionnumbers ATCC ______, ______, ______ and ______, respectively. Anothervector useful for expressing an albumin fusion protein in yeast thepSAC35 vector which is described in Sleep et al., BioTechnology 8:42(1990) which is hereby incorporated by reference in its entirety.

[0148] A variety of methods have been developed to operably link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

[0149] Synthetic linkers containing one or more restriction sitesprovide an alternative method of joining the DNA segment to vectors. TheDNA segment, generated by endonuclease restriction digestion, is treatedwith bacteriophage T4 DNA polymerase or E. coli DNA polymerase I,enzymes that remove protruding, γ-single-stranded termini with their 3′5′-exonucleolytic activities, and fill in recessed 3′-ends with theirpolymerizing activities.

[0150] The combination of these activities therefore generatesblunt-ended DNA segments. The blunt-ended segments are then incubatedwith a large molar excess of linker molecules in the presence of anenzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacteriophage T4 DNA ligase. Thus, the products ofthe reaction are DNA segments carrying polymeric linker sequences attheir ends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

[0151] Synthetic linkers containing a variety of restrictionendonuclease sites are commercially available from a number of sourcesincluding International Biotechnologies Inc, New Haven, Conn., USA.

[0152] A desirable way to modify the DNA in accordance with theinvention, if, for example, HA variants are to be prepared, is to usethe polymerase chain reaction as disclosed by Saiki et al. (1988)Science 239, 487-491. In this method the DNA to be enzymaticallyamplified is flanked by two specific oligonucleotide primers whichthemselves become incorporated into the amplified DNA. The specificprimers may contain restriction endonuclease recognition sites which canbe used for cloning into expression vectors using methods known in theart.

[0153] Exemplary genera of yeast contemplated to be useful in thepractice of the present invention as hosts for expressing the albuminfusion proteins are Pichia (formerly classified as Hansenula),Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis,Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen,Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola,Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium,Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like.Preferred genera are those selected from the group consisting ofSaccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia andTorulaspora. Examples of Saccharomyces spp. are S. cerevisiae, S.italicus and S. rouxii.

[0154] Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K.marxianus. A suitable Torulaspora species is T. delbrueckii. Examples ofPichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P.anomala (formerly H. anomala) and P. pastoris. Methods for thetransformation of S. cerevisiae are taught generally in EP 251 744, EP258 067 and WO 90/01063, all of which are incorporated herein byreference.

[0155] Preferred exemplary species of Saccharomyces include S.cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces rouxii.Preferred exemplary species of Kluyveromyces include K. fragilis and K.lactis. Preferred exemplary species of Hansenula include H. polymorpha(now Pichia angusta), H. anomala (now Pichia anomala), and Pichiacapsulata. Additional preferred exemplary species of Pichia include P.pastoris. Preferred exemplary species of Aspergillus include A. nigerand A. nidulans. Preferred exemplary species of Yarrowia include Y.lipolytica. Many preferred yeast species are available from the ATCC.For example, the following preferred yeast species are available fromthe ATCC and are useful in the expression of albumin fusion proteins:Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant(ATCC Accession No. 4022731); Saccharomyces cerevisiae Hansen,teleomorph strain BY4743 hsp150 mutant (ATCC Accession No. 4021266);Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 pmt1 mutant(ATCC Accession No. 4023792); Saccharomyces cerevisiae Hansen,teleomorph (ATCC Accession Nos. 20626; 44773; 44774; and 62995);Saccharomyces diastaticus Andrews et Gilliland ex van der Walt,teleomorph (ATCC Accession No. 62987); Kluyveromyces lactis (Dombrowski)van der Walt, teleomorph (ATCC Accession No. 76492); Pichia angusta(Teunisson et al.) Kurtzman, teleomorph deposited as Hansenulapolymorpha de Morais et Maia, teleomorph (ATCC Accession No. 26012);Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 9029);Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 16404).Aspergillus nidulans (Eidam) Winter, anamorph (ATCC Accession No.48756); and Yarrowia lipolytica (Wickerham et al.) van der Wait et vonArx, teleomorph (ATCC Accession No. 201847).

[0156] Suitable promoters for S. cerevisiae include those associatedwith the PGKI gene, GAL1 or GAL10 genes, CYCI, PHO5, TRPI, ADHI, ADH2,the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [amating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDIpromoter, and hybrid promoters involving hybrids of parts of 5′regulatory regions with parts of 5′ regulatory regions of otherpromoters or with upstream activation sites (e.g. the promoter ofEP-A-258 067).

[0157] Convenient regulatable promoters for use in Schizosaccharomycespombe are the thiamine-repressible promoter from the nmt gene asdescribed by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and theglucose repressible jbpl gene promoter as described by Hoffman & Winston(1990) Genetics 124, 807-816.

[0158] Methods of transforming Pichia for expression of foreign genesare taught in, for example, Cregg et al. (1993), and various Phillipspatents (e.g. U.S. Pat. No. 4,857,467, incorporated herein byreference), and Pichia expression kits are commercially available fromInvitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego,Calif. Suitable promoters include AOX1 and AOX2. Gleeson et al. (1986)J. Gen. Microbiol. 132, 3459-3465 include information on Hansenulavectors and transformation, suitable promoters being MOX1 and FMD1;whilst EP 361 991, Fleer et al. (1991) and other publications fromRhone-Poulenc Rorer teach how to express foreign proteins inKluyveromyces spp., a suitable promoter being PGKI.

[0159] The transcription termination signal is preferably the 3′flanking sequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3′ flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, i.e. may correspond to the promoter.Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae ADHI gene is preferred.

[0160] The desired albumin fusion protein may be initially expressedwith a secretion leader sequence, which may be any leader effective inthe yeast chosen. Leaders useful in S. cerevisiae include that from themating factor α polypeptide (MF α-1) and the hybrid leaders of EP-A-387319. Such leaders (or signals) are cleaved by the yeast before themature albumin is released into the surrounding medium. Further suchleaders include those of S. cerevisiae invertase (SUC2) disclosed in JP62-096086 (granted as 911036516), acid phosphatase (PH05), thepre-sequence of MFα-1, 0 glucanase (BGL2) and killer toxin; S.diastaticus glucoarnylase 11; S. carlsbergensis α-galactosidase (MEL1);K. lactis killer toxin; and Candida glucoarnylase.

[0161] Additional Methods of Recombinant and Synthetic Production ofAlbumin Fusion Proteins

[0162] The present invention also relates to vectors containing apolynucleotide encoding an albumin fusion protein of the presentinvention, host cells, and the production of albumin fusion proteins bysynthetic and recombinant techniques. The vector may be, for example, aphage, plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

[0163] The polynucleotides encoding albumin fusion proteins of theinvention may be joined to a vector containing a selectable marker forpropagation in a host. Generally, a plasmid vector is introduced in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. If the vector is a virus, it may be packaged invitro using an appropriate packaging cell line and then transduced intohost cells.

[0164] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0165] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418, glutamine synthase, or neomycin resistance foreukaryotic cell culture, and tetracycline, kanamycin or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS,NSO, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0166] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPA0815 (all available from Invitrogen, Caribad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0167] In one embodiment, polynucleotides encoding an albumin fusionprotein of the invention may be fused to signal sequences which willdirect the localization of a protein of the invention to particularcompartments of a prokaryotic or eukaryotic cell and/or direct thesecretion of a protein of the invention from a prokaryotic or eukaryoticcell. For example, in E. coli, one may wish to direct the expression ofthe protein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the albumin fusion proteins ofthe invention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence., MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, polynucleotides albumin fusionproteins of the invention may be fused to the pelB pectate lyase signalsequence to increase the efficiency of expression and purification ofsuch polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos.5,576,195 and 5,846,818, the contents of which are herein incorporatedby reference in their entireties.

[0168] Examples of signal peptides that may be fused to an albuminfusion protein of the invention in order to direct its secretion inmammalian cells include, but are not limited to, the MPIF-1 signalsequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134),the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:34), anda consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:35). Asuitable signal sequence that may be used in conjunction withbaculoviral expression systems is the gp67 signal sequence (e.g., aminoacids 1-19 of GenBank Accession Number AAA72759).

[0169] Vectors which use glutamine synthase (GS) or DHFR as theselectable markers can be amplified in the presence of the drugsmethionine sulphoximine or methotrexate, respectively. An advantage ofglutamine synthase based vectors are the availabilty of cell lines(e.g., the murine myeloma cell line, NSO) which are glutamine synthasenegative. Glutamine synthase expression systems can also function inglutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO)cells) by providing additional inhibitor to prevent the functioning ofthe endogenous gene. A glutamine synthase expression system andcomponents thereof are detailed in PCT publications: WO87/04462;WO86/05807; WO89101036; WO89/10404; and WO91/06657, which are herebyincorporated in their entireties by reference herein. Additionally,glutamine synthase expression vectors can be obtained from LonzaBiologics. Inc. (Portsmouth, N.H.). Expression and production ofmonoclonal antibodies using a GS expression system in murine myelomacells is described in Bebbington et al., Bio/technology 10:169(1992) andin Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are hereinincorporated by reference.

[0170] The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. A host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed.

[0171] Introduction of the nucleic acids and nucleic acid constructs ofthe invention into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986). It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

[0172] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., the coding sequencecorresponding to a Therapeutic protein may be replaced with an albuminfusion protein corresponding to the Therapeutic protein), and/or toinclude genetic material (e.g., heterologous polynucleotide sequencessuch as for example, an albumin fusion protein of the inventioncorresponding to the Therapeutic protein may be included). The geneticmaterial operably associated with the endogenous polynucleotide mayactivate, alter, and/or amplify endogenous polynucleotides.

[0173] In addition, techniques known in the art may be used to operablyassociate heterologous polynucleotides (e.g., polynucleotides encodingan albumin protein, or a fragment or variant thereof) and/orheterologous control regions (e.g., promoter and/or enhancer) withendogenous polynucleotide sequences encoding a Therapeutic protein viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication Number WO 96/29411;International Publication Number WO 94/12650; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989), the disclosures of each of which are incorporated byreference in their entireties).

[0174] Albumin fusion proteins of the invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, hydrophobic charge interactionchromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0175] In preferred embodiments the albumin fusion proteins of theinvention are purified using Anion Exchange Chromatography including,but not limited to, chromatography on Q-sepharose, DEAE sepharose, porosHQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE,Resource/Source Q and DEAE, Fractogel Q and DEAE columns.

[0176] In specific embodiments the albumin fusion proteins of theinvention are purified using Cation Exchange Chromatography including,but not limited to, SP-sepharose, CM sepharose, poros HS, poros CM,Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CMcolumns and their equivalents and comparables.

[0177] In specific embodiments the albumin fusion proteins of theinvention are purified using Hydrophobic Interaction Chromatographyincluding, but not limited to, Phenyl, Butyl, Methyl, Octyl,Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Hexyl, ToyopearlPhenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl,Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexylcolumns and their equivalents and comparables.

[0178] In specific embodiments the albumin fusion proteins of theinvention are purified using Size Exclusion Chromatography including,but not limited to, sepharose S100, S200, S300, superdex resin columnsand their equivalents and comparables.

[0179] In specific embodiments the albumin fusion proteins of theinvention are purified using Affinity Chromatography including, but notlimited to, Mimetic Dye affinity, peptide affinity and antibody affinitycolumns that are selective for either the HSA or the “fusion target”molecules.

[0180] In preferred embodiments albumin fusion proteins of the inventionare purified using one or more Chromatography methods listed above. Inother preferred embodiments, albumin fusion proteins of the inventionare purified using one or more of the following Chromatography columns,Q sepharose FF column, SP Sepharose FF column, Q Sepharose HighPerformance Column, Blue Sepharose FF column, Blue Column, PhenylSepharose FF column, DEAE Sepharose FF, or Methyl Column.

[0181] Additionally, albumin fusion proteins of the invention may bepurified using the process described in International Publication No.WO00/44772 which is herein incorporated by reference in its entirety.One of skill in the art could easily modify the process describedtherein for use in the purification of albumin fusion proteins of theinvention.

[0182] Albumin fusion proteins of the present invention may be recoveredfrom: products of chemical synthetic procedures; and products producedby recombinant techniques from a prokaryotic or eukaryotic host,including, for example, bacterial, yeast, higher plant, insect, andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, albumin fusionproteins of the invention may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0183] In one embodiment, the yeast Pichia pastoris is used to expressalbumin fusion proteins of the invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. SeeEllis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0184] In one example, the plasmid vector pPIC9K is used to express DNAencoding an albumin fusion protein of the invention, as set forthherein, in a Pichea yeast system essentially as described in “PichiaProtocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg,eds. The Humana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a polypeptide of the invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

[0185] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

[0186] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide encoding analbumin fusion protein of the present invention, may be achieved bycloning the heterologous polynucleotide of the invention into anexpression vector such as, for example, pGAPZ or pGAPZalpha, and growingthe yeast culture in the absence of methanol.

[0187] In addition, albumin fusion proteins of the invention can bechemically synthesized using techniques known in the art (e.g., seeCreighton, 1983, Proteins: Structures and Molecular Principles, W. H.Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111(1984)). For example, a polypeptide corresponding to a fragment of apolypeptide can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0188] The invention encompasses albumin fusion proteins of the presentinvention which are differentially modified during or after translation,e.g., by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, etc.Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited, to specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄;acetylation, formylation, oxidation, reduction; metabolic synthesis inthe presence of tunicamycin; etc.

[0189] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The albumin fusion proteins may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.

[0190] Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, and ⁹⁷Ru.

[0191] In specific embodiments, albumin fusion proteins of the presentinvention or fragments or variants thereof are attached to macrocyclicchelators that associate with radiometal ions, including but not limitedto, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³, ¹⁸⁸Sm, to polypeptides. In a preferredembodiment, the radiometal ion associated with the macrocyclic chelatorsis ¹¹¹In. In another preferred embodiment, the radiometal ion associatedwith the macrocyclic chelator is ⁹⁰Y. In specific embodiments, themacrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, DOTA is attached to an antibody of theinvention or fragment thereof via linker molecule. Examples of linkermolecules useful for conjugating DOTA to a polypeptide are commonlyknown in the art—see, for example, DeNardo et al., Clin Cancer Res.4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); whichare hereby incorporated by reference in their entirety.

[0192] As mentioned, the albumin fusion proteins of the invention may bemodified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques which are well knownin the art. It will be appreciated that the same type of modificationmay be present in the same or varying degrees at several sites in agiven polypeptide. Polypeptides of the invention may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic polypeptidesmay result from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

[0193] Albumin fusion proteins of the invention and antibodies that binda Therapeutic protein or fragments or variants thereof can be fused tomarker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

[0194] Further, an albumin fusion protein of the invention may beconjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxinor cytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, coichicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0195] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors. Techniques for conjugating suchtherapeutic moiety to proteins (e.g., albumin fusion proteins) are wellknown in the art.

[0196] Albumin fusion proteins may also be attached to solid supports,which are particularly useful for immunoassays or purification ofpolypeptides that are bound by, that bind to, or associate with albuminfusion proteins of the invention. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

[0197] Albumin fusion proteins, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

[0198] Also provided by the invention are chemically modifiedderivatives of the albumin fusion proteins of the invention which mayprovide additional advantages such as increased solubility, stabilityand circulating time of the polypeptide, or decreased immunogenicity(see U.S. Pat. No. 4,179,337). The chemical moieties for derivitizationmay be selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The albumin fusion proteins maybe modified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0199] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a Therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0200] As noted above, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), thedisclosures of each of which are incorporated herein by reference.

[0201] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, such as, forexample, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF),herein incorporated by reference; see also Malik et al., Exp. Hematol.20:1028-1035 (1992), reporting pegylation of GM-CSF using tresylchloride. For example, polyethylene glycol may be covalently boundthrough amino acid residues via reactive group, such as a free amino orcarboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

[0202] As suggested above, polyethylene glycol may be attached toproteins via linkage to any of a number of amino acid residues. Forexample, polyethylene glycol can be linked to proteins via covalentbonds to lysine, histidine, aspartic acid, glutamic acid, or cysteineresidues. One or more reaction chemistries may be employed to attachpolyethylene glycol to specific amino acid residues (e.g., lysine,histidine, aspartic acid, glutamic acid, or cysteine) of the protein orto more than one type of amino acid residue (e.g., lysine, histidine,aspartic acid, glutamic acid, cysteine and combinations thereof) of theprotein.

[0203] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0204] As indicated above, pegylation of the albumin fusion proteins ofthe invention may be accomplished by any number of means. For example,polyethylene glycol may be attached to the albumin fusion protein eitherdirectly or by an intervening linker. Linkerless systems for attachingpolyethylene glycol to proteins are described in Delgado et al., Crit.Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern.J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No.5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each ofwhich are incorporated herein by reference.

[0205] One system for attaching polyethylene glycol directly to aminoacid residues of proteins without an intervening linker employstresylated MPEG, which is produced by the modification of monmethoxypolyethylene glycol (MPEG) using tresylchloride (CISO₂CH₂CF₃). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the protein. Thus, the inventionincludes protein-polyethylene glycol conjugates produced by reactingproteins of the invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

[0206] Polyethylene glycol can also be attached to proteins using anumber of different intervening linkers. For example, U.S. Pat. No.5,612,460, the entire disclosure of which is incorporated herein byreference, discloses urethane linkers for connecting polyethylene glycolto proteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin International Publication No. WO 98/32466, the entire disclosure ofwhich is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

[0207] The number of polyethylene glycol moieties attached to eachalbumin fusion protein of the invention (i.e., the degree ofsubstitution) may also vary. For example, the pegylated proteins of theinvention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, theaverage degree of substitution within ranges such as 1-3,2-4, 3-5,4-6,5-7,6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17,16-18, 17-19, or 18-20 polyethylene glycol moieties per proteinmolecule. Methods for determining the degree of substitution arediscussed, for example, in Delgado et al., Crit. Rev. Thera. DrugCarrier Sys. 9:249-304 (1992).

[0208] The polypeptides of the invention can be recovered and purifiedfrom chemical synthesis and recombinant cell cultures by standardmethods which include, but are not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and/orpurification.

[0209] The presence and quantity of albumin fusion proteins of theinvention may be determined using ELISA, a well known immunoassay knownin the art. In one ELISA protocol that would be useful fordetecting/quantifying albumin fusion proteins of the invention,comprises the steps of coating an ELISA plate with an anti-human serumalbumin antibody, blocking the plate to prevent non-specific binding,washing the ELISA plate, adding a solution containing the albumin fusionprotein of the invention (at one or more different concentrations),adding a secondary anti-Therapeutic protein specific antibody coupled toa detectable label (as described herein or otherwise known in the art),and detecting the presence of the secondary antibody. In an alternateversion of this protocol, the ELISA plate might be coated with theanti-Therapeutic protein specific antibody and the labeled secondaryreagent might be the anti-human albumin specific antibody.

[0210] Uses of the Polynucleotides

[0211] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0212] The polynucleotides of the present invention are useful toproduce the albumin fusion proteins of the invention. As described inmore detail below, polynucleotides of the invention (encoding albuminfusion proteins) may be used in recombinant DNA methods useful ingenetic engineering to make cells, cell lines, or tissues that expressthe albumin fusion protein encoded by the polynucleotides encodingalbumin fusion proteins of the invention.

[0213] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. Additionalnon-limiting examples of gene therapy methods encompassed by the presentinvention are more thoroughly described elsewhere herein (see, e.g., thesections labeled “Gene Therapy”, and Examples 17 and 18).

[0214] Uses of the Polypeptides

[0215] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0216] Albumin fusion proteins of the invention are useful to provideimmunological probes for differential identification of the tissue(s)(e.g., immunohistochemistry assays such as, for example, ABCimmunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981))or cell type(s) (e.g., immunocytochemistry assays).

[0217] Albumin fusion proteins can be used to assay levels ofpolypeptides in a biological sample using classical immunohistologicalmethods known to those of skill in the art (e.g., see Jalkanen, et al.,J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol.105:3087-3096 (1987)). Other methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable assay labels areknown in the art and include enzyme labels, such as, glucose oxidase;radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In),and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe) fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0218] Albumin fusion proteins of the invention can also be detected invivo by imaging. Labels or markers for in vivo imaging of proteininclude those detectable by X-radiography, nuclear magnetic resonance(NMR) or electron spin relaxtion (ESR). For X-radiography, suitablelabels include radioisotopes such as barium or cesium, which emitdetectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which may be incorporated intothe albumin fusion protein by labeling of nutrients given to a cell lineexpressing the albumin fusion protein of the invention.

[0219] An albumin fusion protein which has been labeled with anappropriate detectable imaging moiety, such as a radioisotope (forexample, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In),and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹¹³Xe), fluorine (¹⁸F,¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or a materialdetectable by nuclear magnetic resonance, is introduced (for example,parenterally, subcutaneously or intraperitoneally) into the mammal to beexamined for immune system disorder. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ^(99m)Tc. The labeled albumin fusion protein will thenpreferentially accumulate at locations in the body (e.g., organs, cells,extracellular spaces or matrices) where one or more receptors, ligandsor substrates (corresponding to that of the Therapeutic protein used tomake the albumin fusion protein of the invention) are located.Alternatively, in the case where the albumin fusion protein comprises atleast a fragment or variant of a Therapeutic antibody, the labeledalbumin fusion protein will then preferentially accumulate at thelocations in the body (e.g., organs, cells, extracellular spaces ormatrices) where the polypeptideslepitopes corresponding to those boundby the Therapeutic antibody (used to make the albumin fusion protein ofthe invention) are located. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982)). The protocols described therein could easily bemodified by one of skill in the art for use with the albumin fusionproteins of the invention.

[0220] In one embodiment, the invention provides a method for thespecific delivery of albumin fusion proteins of the invention to cellsby administering albumin fusion proteins of the invention (e.g.,polypeptides encoded by polynucleotides encoding albumin fusion proteinsof the invention and/or antibodies) that are associated withheterologous polypeptides or nucleic acids. In one example, theinvention provides a method for delivering a Therapeutic protein intothe targeted cell. In another example, the invention provides a methodfor delivering a single stranded nucleic acid (e.g., antisense orribozymes) or double stranded nucleic acid (e.g., DNA that can integrateinto the cell's genome or replicate episomally and that can betranscribed) into the targeted cell.

[0221] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering albumin fusion proteins of the invention in associationwith toxins or cytotoxic prodrugs.

[0222] By “toxin” is meant one or more compounds that bind and activateendogenous cytotoxic effector systems, radioisotopes, holotoxins,modified toxins, catalytic subunits of toxins, or any molecules orenzymes not normally present in or on the surface of a cell that underdefined conditions cause the cell's death. Toxins that may be usedaccording to the methods of the invention include, but are not limitedto, radioisotopes known in the art, compounds such as, for example,antibodies (or complement fixing containing portions thereof) that bindan inherent or induced endogenous cytotoxic effector system, thymidinekinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonasexotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweedantiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includesa cytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or otherradioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge ⁵⁷Co, ⁶⁵Zn,⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. In a specific embodiment, the invention providesa method for the specific destruction of cells (e.g., the destruction oftumor cells) by administering polypeptides of the invention orantibodies of the invention in association with the radioisotope ⁹⁰Y. Inanother specific embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹¹¹In. In a furtherspecific embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹³¹I.

[0223] Techniques known in the art may be applied to label polypeptidesof the invention. Such techniques include, but are not limited to, theuse of bifunctional conjugating agents (see e.g., U.S. Pat. Nos.5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425;5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003;the contents of each of which are hereby incorporated by reference inits entirety).

[0224] The albumin fusion proteins of the present invention are usefulfor diagnosis, treatment, prevention and/or prognosis of variousdisorders in mammals, preferably humans. Such disorders include, but arenot limited to, those described herein under the section heading“Biological Activities,” below.

[0225] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression level of a certainpolypeptide in cells or body fluid of an individual using an albuminfusion protein of the invention; and (b) comparing the assayedpolypeptide expression level with a standard polypeptide expressionlevel, whereby an increase or decrease in the assayed polypeptideexpression level compared to the standard expression level is indicativeof a disorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0226] Moreover, albumin fusion proteins of the present invention can beused to treat or prevent diseases or conditions such as, for example,neural disorders, immune system disorders, muscular disorders,reproductive disorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor supressor), to activate the activity of a polypeptide (e.g., bybinding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

[0227] In particular, albumin fusion proteins comprising of at least afragment or variant of a Therapeutic antibody can also be used to treatdisease (as described supra, and elsewhere herein). For example,administration of an albumin fusion protein comprising of at least afragment or variant of a Therapeutic antibody can bind, and/orneutralize the polypeptide to which the Therapeutic antibody used tomake the albumin fusion protein immunospecifically binds, and/or reduceoverproduction of the polypeptide to which the Therapeutic antibody usedto make the albumin fusion protein immunospecifically binds. Similarly,administration of an albumin fusion protein comprising of at least afragment or variant of a Therapeutic antibody can activate thepolypeptide to which the Therapeutic antibody used to make the albuminfusion protein immunospecifically binds, by binding to the polypeptidebound to a membrane (receptor).

[0228] At the very least, the albumin fusion proteins of the inventionof the present invention can be used as molecular weight markers onSDS-PAGE gels or on molecular sieve gel filtration columns using methodswell known to those of skill in the art. Albumin fusion proteins of theinvention can also be used to raise antibodies, which in turn may beused to measure protein expression of the Therapeutic protein, albuminprotein, and/or the albumin fusion protein of the invention from arecombinant cell, as a way of assessing transformation of the host cell,or in a biological sample. Moreover, the albumin fusion proteins of thepresent invention can be used to test the biological activitiesdescribed herein.

[0229] Diagnostic Assays

[0230] The compounds of the present invention are useful for diagnosis,treatment, prevention and/or prognosis of various disorders in mammals,preferably humans. Such disorders include, but are not limited to, thosedescribed for each Therapeutic protein in the corresponding row of TableI and herein tinder the section headings “Immune Activity,” “BloodRelated Disorders,” “Hyperproliferative Disorders,” “Renal Disorders,”“Cardiovascular Disorders,” “Respiratory Disorders,” “Anti-AngiogenesisActivity,” “Diseases at the Cellular Level,” “Wound Healing andEpithelial Cell Proliferation,” “Neural Activity and NeurologicalDiseases,” “Endocrine Disorders,” “Reproductive System Disorders,”“Infectious Disease,” “Regeneration,” and/or “GastrointestinalDisorders,” infra.

[0231] For a number of disorders, substantially altered (increased ordecreased) levels of gene expression can be detected in tissues, cellsor bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto a “standard” gene expression level, that is, the expression level intissues or bodily fluids from an individual not having the disorder.Thus, the invention provides a diagnostic method useful during diagnosisof a disorder, which involves measuring the expression level of the geneencoding a polypeptide in tissues, cells or body fluid from anindividual and comparing the measured gene expression level with astandard gene expression level, whereby an increase or decrease in thegene expression level(s) compared to the standard is indicative of adisorder. These diagnostic assays may be performed in vivo or in vitro,such as, for example, on blood samples, biopsy tissue or autopsy tissue.

[0232] The present invention is also useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed gene expression willexperience a worse clinical outcome By “assaying the expression level ofthe gene encoding a polypeptide” is intended qualitatively orquantitatively measuring or estimating the level of a particularpolypeptide (e.g. a polypeptide corresponding to a Therapeutic proteindisclosed in Table 1) or the level of the mRNA encoding the polypeptideof the invention in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level or mRNA level) orrelatively (e.g., by comparing to the polypeptide level or mRNA level ina second biological sample). Preferably, the polypeptide expressionlevel or mRNA level in the first biological sample is measured orestimated and compared to a standard polypeptide level or mRNA level,the standard being taken from a second biological sample obtained froman individual not having the disorder or being determined by averaginglevels from a population of individuals not having the disorder. As willbe appreciated in the art, once a standard polypeptide level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

[0233] By “biological sample” is intended any biological sample obtainedfrom an individual, cell line, tissue culture, or other sourcecontaining polypeptides of the invention (including portions thereof) ormRNA. As indicated, biological samples include body fluids (such assera, plasma, urine, synovial fluid and spinal fluid) and tissue sourcesfound to express the full length or fragments thereof of a polypeptideor mRNA. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0234] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the polypeptides of the invention are then assayed usingany appropriate method. These include Northern blot analysis, S1nuclease mapping, the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction(RT-PCR), and reverse transcription in combination with the ligase chainreaction (RT-LCR).

[0235] The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of polypeptidesthat bind to, are bound by, or associate with albumin fusion proteins ofthe invention, in a biological sample (e.g., cells and tissues),including determination of normal and abnormal levels of polypeptides.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting abnormal expression of polypeptides that bind to, arebound by, or associate with albumin fusion proteins compared to normalcontrol tissue samples may be used to detect the presence of tumors.Assay techniques that can be used to determine levels of a polypeptidethat bind to, are bound by, or associate with albumin fusion proteins ofthe present invention in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Assaying polypeptide levels in a biological sample can occur using anyart-known method.

[0236] Assaying polypeptide levels in a biological sample can occurusing a variety of techniques. For example, polypeptide expression intissues can be studied with classical immunohistological methods(Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., etal., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful fordetecting polypeptide gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase, and radioisotopes, such asiodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0237] The tissue or cell type to be analyzed will generally includethose which are known, or suspected, to express the gene of interest(such as, for example, cancer). The protein isolation methods employedherein may, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cellsthat could be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thegene.

[0238] For example, albumin fusion proteins may be used toquantitatively or qualitatively detect the presence of polypeptides thatbind to, are bound by, or associate with albumin fusion proteins of thepresent invention. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled albuminfusion protein coupled with light microscopic, flow cytometric, orfluorimetric detection.

[0239] In a preferred embodiment, albumin fusion proteins comprising atleast a fragment or variant of an antibody that immunospecifically bindsat least a Therapeutic protein disclosed herein (e.g., the Therapeuticproteins disclosed in Table 1) or otherwise known in the art may be usedto quantitatively or qualitatively detect the presence of gene productsor conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody coupled with light microscopic, flowcytometric, or fluorimetric detection.

[0240] The albumin fusion proteins of the present invention may,additionally, be employed histologically, as in immunofluorescence,immunoelectron microscopy or non-immunological assays, for in situdetection of polypeptides that bind to, are bound by, or associate withan albumin fusion protein of the present invention. In situ detectionmay be accomplished by removing a histological specimen from a patient,and applying thereto a labeled antibody or polypeptide of the presentinvention. The albumin fusion proteins are preferably applied byoverlaying the labeled albumin fusion proteins onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the polypeptides that bind to, are bound by, orassociate with albumin fusion proteins, but also its distribution in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

[0241] Immunoassays and non-immunoassays that detect polypeptides thatbind to, are bound by, or associate with albumin fusion proteins willtypically comprise incubating a sample, such as a biological fluid, atissue extract, freshly harvested cells, or lysates of cells which havebeen incubated in cell culture, in the presence of a detectably labeledantibody capable of binding gene products or conserved variants orpeptide fragments thereof, and detecting the bound antibody by any of anumber of techniques well-known in the art.

[0242] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled albumin fusion protein of the invention. The solid phase supportmay then be washed with the buffer a second time to remove unboundantibody or polypeptide. Optionally the antibody is subsequentlylabeled. The amount of bound label on solid support may then be detectedby conventional means.

[0243] By “solid phase support or carrier” is intended any supportcapable of binding a polypeptide (e.g., an albumin fusion protein, orpolypeptide that binds, is bound by, or associates with an albuminfusion protein of the invention.) Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding to apolypeptide. Thus, the support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube, or theexternal surface of a rod. Alternatively, the surface may be flat suchas a sheet, test strip, etc. Preferred supports include polystyrenebeads. Those skilled in the art will know many other suitable carriersfor binding antibody or antigen, or will be able to ascertain the sameby use of routine experimentation.

[0244] The binding activity of a given lot of albumin fusion protein maybe determined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

[0245] In addition to assaying polypeptide levels in a biological sampleobtained from an individual, polypeptide can also be detected in vivo byimaging. For example, in one embodiment of the invention, albumin fusionproteins of the invention are used to image diseased or neoplasticcells.

[0246] Labels or markers for in vivo imaging of albumin fusion proteinsof the invention include those detectable by X-radiography, NMR, MRI,CAT-scans or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the albumin fusion protein bylabeling of nutrients of a cell line (or bacterial or yeast strain)engineered.

[0247] Additionally, albumin fusion proteins of the invention whosepresence can be detected, can be administered. For example, albuminfusion proteins of the invention labeled with a radio-opaque or otherappropriate compound can be administered and visualized in vivo, asdiscussed, above for labeled antibodies. Further, such polypeptides canbe utilized for in vitro diagnostic procedures.

[0248] A polypeptide-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorder. Itwill be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled albuminfusion protein will then preferentially accumulate at the locations inthe body which contain a polypeptide or other substance that binds to,is bound by or associates with an albumin fusion protein of the presentinvention. In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

[0249] One of the ways in which an albumin fusion protein of the presentinvention can be detectably labeled is by linking the same to a reporterenzyme and using the linked product in an enzyme immunoassay (EIA)(Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978,Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.); Voller et al., J. Clin. Pathol31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton,Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, KgakuShoin, Tokyo). The reporter enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Reporter enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for thereporter enzyme. Detection may also be accomplished by visual comparisonof the extent of enzymatic reaction of a substrate in comparison withsimilarly prepared standards.

[0250] Albumin fusion proteins may also be radiolabelled and used in anyof a variety of other immunoassays. For example, by radioactivelylabeling the albumin fusion proteins, it is possible to the use thealbumin fusion proteins in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

[0251] It is also possible to label the albumin fusion proteins with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

[0252] The albumin fusion protein can also be detectably labeled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthamideseries. These metals can be attached to the antibody using such metalchelating groups as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0253] The albumin fusion proteins can also can be detectably labeled bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged albumin fusion protein is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

[0254] Likewise, a bioluminescent compound may be used to label albuminfusion proteins of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0255] Transgenic Organisms

[0256] Transgenic organisms that express the albumin fusion proteins ofthe invention are also included in the invention. Transgenic organismsare genetically modified organisms into which recombinant, exogenous orcloned genetic material has been transferred. Such genetic material isoften referred to as a transgene. The nucleic acid sequence of thetransgene may include one or more transcriptional regulatory sequencesand other nucleic acid sequences such as introns, that may be necessaryfor optimal expression and secretion of the encoded protein. Thetransgene may be designed to direct the expression of the encodedprotein in a manner that facilitates its recovery from the organism orfrom a product produced by the organism, e.g. from the milk, blood,urine, eggs, hair or seeds of the organism. The transgene may consist ofnucleic acid sequences derived from the genome of the same species or ofa different species than the species of the target animal. The transgenemay be integrated either at a locus of a genome where that particularnucleic acid sequence is not otherwise normally found or at the normallocus for the transgene.

[0257] The term “germ cell line transgenic organism” refers to atransgenic organism in which the genetic alteration or geneticinformation was introduced into a germ line cell, thereby conferring theability of the transgenic organism to transfer the genetic informationto offspring. If such offspring in fact possess some or all of thatalteration or genetic information, then they too are transgenicorganisms. The alteration or genetic information may be foreign to thespecies of organism to which the recipient belongs, foreign only to theparticular individual recipient, or may be genetic information alreadypossessed by the recipient. In the last case, the altered or introducedgene may be expressed differently than the native gene.

[0258] A transgenic organism may be a transgenic animal or a transgenicplant. Transgenic animals can be produced by a variety of differentmethods including transfection, electroporation, microinjection, genetargeting in embryonic stem cells and recombinant viral and retroviralinfection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307;Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997)Surg. Oncol. 6(2)₉₉-110; Tuan (ed.), Recombinant Gene ExpressionProtocols, Methods in Molecular Biology No. 62, Humana Press (1997)).The method of introduction of nucleic acid fragments into recombinationcompetent mammalian cells can be by any method which favorsco-transformation of multiple nucleic acid molecules. Detailedprocedures for producing transgenic animals are readily available to oneskilled in the art, including the disclosures in U.S. Pat. No. 5,489,743and U.S. Pat. No. 5,602,307.

[0259] A number of recombinant or transgenic mice have been produced,including those which express an activated oncogene sequence (U.S. Pat.No. 4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915);lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat.No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No.5,723,719); express at least one human gene which participates in bloodpressure control (U.S. Pat. No. 5,731,489); display greater similarityto the conditions existing in naturally occurring Alzheimer's disease(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellularadhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene(Clutter et al. (1996) Genetics 143(4): 1753-1760); or, are capable ofgenerating a fully human antibody response (McCarthy (1997) The Lancet349(9049):405).

[0260] While mice and rats remain the animals of choice for mosttransgenic experimentation, in some instances it is preferable or evennecessary to use alternative animal species. Transgenic procedures havebeen successfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev.46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617;Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al. (1997)Science 278(5346):2130-2133; and Amoah (1997) J. Animal Science75(2):578-585).

[0261] To direct the secretion of the transgene-encoded protein of theinvention into the milk of transgenic mammals, it may be put under thecontrol of a promoter that is preferentially activated in mammaryepithelial cells. Promoters that control the genes encoding milkproteins are preferred, for example the promoter for casein, betalactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio(1992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology7:487-492; Gorton et al. (1987) BioTechnology 5:1183-1187; and Soulieret al. (1992) FEBS Letts. 297:13). The transgenic mammals of choicewould produce large volumes of milk and have long lactating periods, forexample goats, cows, camels or sheep.

[0262] An albumin fusion protein of the invention can also be expressedin a transgenic plant, e.g. a plant in which the DNA transgene isinserted into the nuclear or plastidic genome. Plant transformationprocedures used to introduce foreign nucleic acids into plant cells orprotoplasts are known in the art (e.g., see Example 19). See, ingeneral, Methods in Enzymology Vol. 153 (“Recombinant DNA Part D”) 1987,Wu and Grossman Eds., Academic Press and European Patent Application EP693554. Methods for generation of genetically engineered plants arefurther described in U.S. Pat. No. 5,283,184, U.S. Pat. No. 5, 482,852,and European Patent Application EP 693 554, all of which are herebyincorporated by reference.

[0263] Pharmaceutical or Therapeutic Compositions

[0264] The albumin fusion proteins of the invention or formulationsthereof may be administered by any conventional method includingparenteral (e.g. subcutaneous or intramuscular) injection or intravenousinfusion. The treatment may consist of a single dose or a plurality ofdoses over a period of time.

[0265] While it is possible for an albumin fusion protein of theinvention to be administered alone, it is preferable to present it as apharmaceutical formulation, together with one or more acceptablecarriers. The carrier(s) must be “acceptable” in the sense of beingcompatible with the albumin fusion protein and not deleterious to therecipients thereof. Typically, the carriers will be water or salinewhich will be sterile and pyrogen free. Albumin fusion proteins of theinvention are particularly well suited to formulation in aqueouscarriers such as sterile pyrogen free water, saline or other isotonicsolutions because of their extended shelf-life in solution. Forinstance, pharmaceutical compositions of the invention may be formulatedwell in advance in aqueous form, for instance, weeks or months or longertime periods before being dispensed.

[0266] For example, wherein the Therapeutic protein is hGH, EPO,alpha-IFN or beta-IFN, formulations containing the albumin fusionprotein may be prepared taking into account the extended shelf-life ofthe albumin fusion protein in aqueous formulations. As exhibited inTable 2, most Therapeutic proteins are unstable with short shelf-livesafter formulation with an aqueous carrier. As discussed above, theshelf-life of many of these Therapeutic proteins are markedly increasedor prolonged after fusion to HA. TABLE 2 Tradename, Storage Conditionsof Protein Manufacturer Route Formulation Non-Fusion Protein Interferon,Roferon-A, sc sol_n 4-8° C. alpha-2a Hoffmann-LaRoche im (vial orpre-filled syringe) Interferon, Intron-A, iv sc im sol_n; 4-8° C.alpha-2b Schering Plough powder + dil. (all preps, before and afterdilution) COMBO Rebetron (Intron-A + po + Rebetol capsule + Interferonalpha- Rebetol) sc Intron-A injection 2b + Schering Plough RibavirinInterferon, Infergen sc sol_n 4-8° C. Alphacon-1 Amgen Interferon,Wellferon, sc sol_n 4-8° C. alpha-n1, Wellcome im (with albumin _asLympho- stablizer_) blastoid Interferon, Avonex, im powder + dil. 4-8°C. beta-1a Biogen (with albumin) (before and after dilution) (Use within3-6 h of reconstitution) Rebif, sc sol_n, Ares-Serono in pre-filledsyringe (Europe only) Interferon, Betaseron, sc powder + dil. 4-8° C.beta-1b Chiron (with albumin) (before and after dilution) (Europe:Betaferon) (Use within 3 h of reconstitution) Single use vials.Interferon, Actimmune, sc 4-8° C. Gamma-1b InterMune (before and afterdilution) Pharmaceuticals (Use within 3 h of reconstitution). GrowthGenotropin, powder/dil cartridges 4-8° C. Hormone Pharmacia Upjohn(single or multi-use); (before and after dilution); (somatropin) singleuse MiniQuick single use MiniQuick injector Delivery Device should berefrigerated until use. Humatrope, sc powder + dil. 4-8° C. Eli Lilly im(Vial or pen cartridge) (before and after dilution) (Use vials within 25h, cartridges within 28 d, of reconstitution). Norditropin, Novo NordiskPharmaceuticals Nutropin, sc powder + dil. 4-8° C. Genentech (stable for14 d after dil_n) (all preps, before and after dilution) Nutropin AQ, scsol_n 4-8° C. Genentech (Stable for 28 d after 1st use) Nutropin Depot.sc microsphere 4-8° C. Genentech suspension as Single use pkges. Dosepowder + dil. 1-2x/month (ProLease micro-encapsulation technol.) Saizen,sc powder + dil. Powder _should be stored (Serono) im at Rm Temp_. Afterreconstitution store 4- 8° C. for up to 14 d. Serostim, Powder _shouldbe stored Serono at Rm Temp_. After reconstitution store in 4- 8° C. forup to 14 d. hGH, with Protropin, sc powder + dil. 4-8° C. N-term. MetGenentech im (all preps, before and (somatrem) after dilution)Erythropoietin Epogen, iv sol_n 4-8° C. (Epoetin alfa) Amgen sc (usewithin 21 d of first use) (Single & multi-dose vials) Procrit, iv sol_n4-8° C. Amgen sc (use within 21 d of first use) (Single & multi-dosevials)

[0267] In instances where aerosol administration is appropriate, thealbumin fusion proteins of the invention can be formulated as aerosolsusing standard procedures. The term “aerosol” includes any gas-bornesuspended phase of an albumin fusion protein of the instant inventionwhich is capable of being inhaled into the bronchioles or nasalpassages. Specifically, aerosol includes a gas-borne suspension ofdroplets of an albumin fusion protein of the instant invention, as maybe produced in a metered dose inhaler or nebulizer, or in a mistsprayer. Aerosol also includes a dry powder composition of a compound ofthe instant invention suspended in air or other carrier gas, which maybe delivered by insufflation from an inhaler device, for example. SeeGanderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood(1987); Gonda (1990) Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313; and Raeburn et al,. (1992) Pharmacol. Toxicol.Methods 27:143-159.

[0268] The formulations of the invention are also typicallynon-immunogenic, in part, because of the use of the components of thealbumin fusion protein being derived from the proper species. Forinstance, for human use, both the Therapeutic protein and albuminportions of the albumin fusion protein will typically be human. In somecases, wherein either component is non human-derived, that component maybe humanized by substitution of key amino acids so that specificepitopes appear to the human immune system to be human in nature ratherthan foreign.

[0269] The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Such methods include the step of bringing into association thealbumin fusion protein with the carrier that constitutes one or moreaccessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product. Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation appropriate for the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampules, vials or syringes, and may be stored in a freeze-dried(lyophilised) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders. Dosage formulations may contain the Therapeuticprotein portion at a lower molar concentration or lower dosage comparedto the non-fused standard formulation for the Therapeutic protein giventhe extended serum half-life exhibited by many of the albumin fusionproteins of the invention.

[0270] As an example, when an albumin fusion protein of the inventioncomprises growth hormone as one or more of the Therapeutic proteinregions, the dosage form can be calculated on the basis of the potencyof the albumin fusion protein relative to the potency of hGH, whiletaking into account the prolonged serum half-life and shelf-life of thealbumin fusion proteins compared to that of native hGH. Growth hormoneis typically administered at 0.3 to 30.0 IU/kg/week, for example 0.9 to12.0 IU/kg/week, given in three or seven divided doses for a year ormore. In an albumin fusion protein consisting of full length HA fused tofull length GH, an equivalent dose in terms of units would represent agreater weight of agent but the dosage frequency can be reduced, forexample to twice a week, once a week or less.

[0271] Formulations or compositions of the invention may be packagedtogether with, or included in a kit with, instructions or a packageinsert referring to the extended shelf-life of the albumin fusionprotein component. For instance, such instructions or package insertsmay address recommended storage conditions, such as time, temperatureand light, taking into account the extended or prolonged shelf-life ofthe albumin fusion proteins of the invention. Such instructions orpackage inserts may also address the particular advantages of thealbumin fusion proteins of the inventions, such as the ease of storagefor formulations that may require use in the field, outside ofcontrolled hospital, clinic or office conditions. As described above,formulations of the invention may be in aqueous form and may be storedunder less than ideal circumstances without significant loss oftherapeutic activity.

[0272] Albumin fusion proteins of the invention can also be included innutraceuticals. For instance, certain albumin fusion proteins of theinvention may be administered in natural products, including milk ormilk product obtained from a transgenic mammal which expresses albuminfusion protein. Such compositions can also include plant or plantproducts obtained from a transgenic plant which expresses the albuminfusion protein. The albumin fusion protein can also be provided inpowder or tablet form, with or without other known additives, carriers,fillers and diluents. Nutraceuticals are described in Scott Hegenhart,Food Product Design, December 1993.

[0273] The invention also provides methods of treatment and/orprevention of diseases or disorders (such as, for example, any one ormore of the diseases or disorders disclosed herein) by administration toa subject of an effective amount of an albumin fusion protein of theinvention or a polynucleotide encoding an albumin fusion protein of theinvention (“albumin fusion polynucleotide”) in a pharmaceuticallyacceptable carrier.

[0274] The albumin fusion protein and/or polynucleotide will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with the albumin fusionprotein and/or polynucleotide alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[0275] As a general proposition, the total pharmaceutically effectiveamount of the albumin fusion protein administered parenterally per dosewill be in the range of about lug/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the albumin fusion protein is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0276] Albumin fusion proteins and/or polynucleotides can be areadministered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray. “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0277] Albumin fusion proteins and/or polynucleotides of the inventionare also suitably administered by sustained-release systems. Examples ofsustained-release albumin fusion proteins and/or polynucleotides areadministered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray. “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion. Additional examples ofsustained-release albumin fusion proteins and/or polynucleotides includesuitable polymeric materials (such as, for example, semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormirocapsules), suitable hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[0278] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[0279] Sustained-release albumin fusion proteins and/or polynucleotidesalso include liposomally entrapped albumin fusion proteins and/orpolynucleotides of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing thealbumin fusion protein and/or polynucleotide are prepared by methodsknown per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA)82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol percent cholesterol, the selected proportionbeing adjusted for the optimal Therapeutic.

[0280] In yet an additional embodiment, the albumin fusion proteinsand/or polynucleotides of the invention are delivered by way of a pump(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)).

[0281] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0282] For parenteral administration, in one embodiment, the albuminfusion protein and/or polynucleotide is formulated generally by mixingit at the desired degree of purity, in a unit dosage injectable form(solution suspension, or emulsion), with a pharmaceutically acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious tothe Therapeutic.

[0283] Generally, the formulations are prepared by contacting thealbumin fusion protein and/or polynucleotide uniformly and intimatelywith liquid carriers or finely divided solid carriers or both. Then, ifnecessary the product is shaped into the desired formulation. Preferablythe carrier is a parenteral carrier, more preferably a solution that isisotonic with the blood of the recipient. Examples of such carriervehicles include water, saline, Ringer's solution, and dextrosesolution. Non-aqueous vehicles such as fixed oils and ethyl oleate arealso useful herein, as well as liposomes.

[0284] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0285] The albumin fusion protein is typically formulated in suchvehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the useof certain of the foregoing excipients, carriers, or stabilizers willresult in the formation of polypeptide salts.

[0286] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Albumin fusionproteins and/or polynucleotides generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

[0287] Albumin fusion proteins and/or polynucleotides ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-ml vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusionprotein and/or polynucleotide solution, and the resulting mixture islyophilized. The infusion solution is prepared by reconstituting thelyophilized albumin fusion protein and/or polynucleotide usingbacteriostatic Water-for-Injection.

[0288] In a specific and preferred embodiment, the Albumin fusionprotein formulations comprises 0.01 M sodium phosphate, 0.15 mM sodiumchloride, 0.16 micromole sodium octanoate/milligram of fusion protein,15 micrograms/milliliter polysorbate 80, pH 7.2. In another specific andpreferred embodiment, the Albumin fusion protein formulations consists0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodiumoctanoate/milligram of fusion protein, 15 micrograms/milliliterpolysorbate 80, pH 7.2. The pH and buffer are chosen to matchphysiological conditions and the salt is added as a tonicifier. Sodiumoctanoate has been chosen due to its reported ability to increase thethermal stability of the protein in solution. Finally, polysorbate hasbeen added as a generic surfactant, which lowers the surface tension ofthe solution and lowers non-specific adsorption of the albumin fusionprotein to the container closure system.

[0289] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the albumin fusion proteins and/or polynucleotides of theinvention. Associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In addition, the albumin fusion proteins and/orpolynucleotides may be employed in conjunction with other therapeuticcompounds.

[0290] The albumin fusion proteins and/or polynucleotides of theinvention may be administered alone or in combination with adjuvants.Adjuvants that may be administered with the albumin fusion proteinsand/or polynucleotides of the invention include, but are not limited to,alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparationsof Corynebacterium parvum. In a specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are administered incombination with alum. In another specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, Monophosphoryl lipid immunomodulator,AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, andVirosomal adjuvant technology. Vaccines that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0291] The albumin fusion proteins and/or polynucleotides of theinvention may be administered alone or in combination with othertherapeutic agents. Albumin fusion protein and/or polynucleotide agentsthat may be administered in combination with the albumin fusion proteinsand/or polynucleotides of the invention, include but not limited to,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, and/ortherapeutic treatments described below. Combinations may be administeredeither concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

[0292] In one embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with ananticoagulant. Anticoagulants that may be administered with thecompositions of the invention include, but are not limited to, heparin,low molecular weight heparin, warfarin sodium (e.g., COUMADIN®),dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADON™),acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione,phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™),and aspirin. In a specific embodiment, compositions of the invention areadministered in combination with heparin and/or warfarin. In anotherspecific embodiment, compositions of the invention are administered incombination with warfarin. In another specific embodiment, compositionsof the invention are administered in combination with warfarin andaspirin. In another specific embodiment, compositions of the inventionare administered in combination with heparin. In another specificembodiment, compositions of the invention are administered incombination with heparin and aspirin.

[0293] In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withthrombolytic drugs. Thrombolytic drugs that may be administered with thecompositions of the invention include, but are not limited to,plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g.,KABIKINASE™), antiresplace (e.g., EMINASE™), tissue plasminogenactivator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™),sauruplase, (Prourokinase, single chain urokinase), and aminocaproicacid (e.g., AMICAR™). In a specific embodiment, compositions of theinvention are administered in combination with tissue plasminogenactivator and aspirin.

[0294] In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withantiplatelet drugs. Antiplatelet drugs that may be administered with thecompositions of the invention include, but are not limited to, aspirin,dipyridamole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).

[0295] In specific embodiments, the use of anti-coagulants, thrombolyticand/or antiplatelet drugs in combination with albumin fusion proteinsand/or polynucleotides of the invention is contemplated for theprevention, diagnosis, and/or treatment of thrombosis, arterialthrombosis, venous thrombosis, thromboembolism, pulmonary embolism,atherosclerosis, myocardial infarction, transient ischemic attack,unstable angina. In specific embodiments, the use of anticoagulants,thrombolytic drugs and/or antiplatelet drugs in combination with albuminfusion proteins and/or polynucleotides of the invention is contemplatedfor the prevention of occulsion of saphenous grafts, for reducing therisk of periprocedural thrombosis as might accompany angioplastyprocedures, for reducing the risk of stroke in patients with atrialfibrillation including nonrheumatic atrial fibrillation, for reducingthe risk of embolism associated with mechanical heart valves and ormitral valves disease. Other uses for the therapeutics of the invention,alone or in combination with antiplatelet, anticoagulant, and/orthrombolytic drugs, include, but are not limited to, the prevention ofocclusions in extracorporeal devices (e.g., intravascular canulas,vascular access shunts in hemodialysis patients, hemodialysis machines,and cardiopulmonary bypass machines).

[0296] In certain embodiments, albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withantiretroviral agents, nucleoside/nucleotide reverse transcriptaseinhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors(NNRTIs), and/or protease inhibitors (PIs). NRTIs that may beadministered in combination with the albumin fusion proteins and/orpolynucleotides of the invention, include, but are not limited to,RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™(zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIV IR™ (lamivudine/3TC),and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administeredin combination with the albumin fusion proteins and/or polynucleotidesof the invention, include, but are not limited to, VIRAMUNE™(nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz).Protease inhibitors that may be administered in combination with thealbumin fusion proteins and/or polynucleotides of the invention,include, but are not limited to. CRIXIVAN™ (indinavir), NORVIR™(ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In aspecific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith albumin fusion proteins and/or polynucleotides of the invention totreat AIDS and/or to prevent or treat HIV infection.

[0297] Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stableadenosine NRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC;structurally related to lamivudine (3TC) but with 3- to 10-fold greateractivity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurallyrelated to lamivudine but retains activity against a substantialproportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir(refused approval for anti-HIV therapy by FDA; Gilead Sciences);PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its activeform is PMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead);DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (relatedto 3TC, with activity against AZT/3TC-resistant virus); GW420867X (GlaxoWellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87(3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of β-L-FD4C and β3-L-FddC (see,International Publication No. WO 98/17281).

[0298] Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potentNNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153,a next generation NNRTI with activity against viruses containing theK103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greateractivity than its predecessor delavirdine and is active against K103Nmutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generationderivatives of efavirenz, designed to be active against viruses with theK103N mutation; DuPont); GW-420867×(has 25-fold greater activity thanHBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDEA (naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(see, International Publication No. WO 99/49830).

[0299] Additional protease inhibitors include LOPINAVIR™ (ABT378/r;Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

[0300] Additional antiretroviral agents include fusion inhibitors/gp41binders. Fusion inhibitors/gp4l binders include T-20 (a peptide fromresidues 643-678 of the HIV gp41 transmembrane protein ectodomain whichbinds to gp41 in its resting state and prevents transformation to thefusogenic state; Trimeris) and T-1249 (a second-generation fusioninhibitor; Trimeris).

[0301] Additional antiretroviral agents include fusioninhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokinereceptor antagonists include CXCR4 antagonists such as AMD 3100 (abicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22(an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140;CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, andTAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycinanalog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokinerecpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may alsoinhibit fusion.

[0302] Additional antiretroviral agents include integrase inhibitors.Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoricacid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

[0303] Additional antiretroviral agents include hydroxyurea-likecompunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor;Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Moleculesfor Health); inosine monophosphate dehydrogenase (IMPDH) inhibitorssucha as VX497 (Vertex); and mycopholic acids such as CellCept(mycophenolate mofetil; Roche).

[0304] Additional antiretroviral agents include inhibitors of viralintegrase, inhibitors of viral genome nuclear translocation such asarylene bis(methylketone) compounds; inhibitors of HIV entry such asAOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes ofRANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zincfinger inhibitors such as dithiane compounds; targets of HIV Tat andRev; and pharmacoenhancers such as ABT-378.

[0305] Other antiretroviral therapies and adjunct therapies includecytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2,PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120,gp120/soluble CD4 complex, Delta JR-FL protein, branched syntheticpeptide derived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(genetically modified CC chemokines targetted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as theanti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9,PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4,the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d,447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-αantibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptoragonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl,3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (see, InternationalPublication No. WO 98/30213); and antioxidants such asγ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

[0306] In a further embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantiviral agent. Antiviral agents that may be administered with thealbumin fusion proteins and/or polynucleotides of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0307] In other embodiments, albumin fusion proteins and/orpolynucleotides of the invention may be administered in combination withanti-opportunistic infection agents. Anti-opportunistic agents that maybe administered in combination with the albumin fusion proteins and/orpolynucleotides of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUT™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteinsand/or polynucleotides of the invention are used in any combination withTRIMETHOPRIM-SULFAMETHOAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, albumin fusion proteins and/or polynucleotides of theinvention are used in any combination with ISONIAZID™, RIFAMPIN™,PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or preventan opportunistic Mycobacterium avium complex infection. In anotherspecific embodiment, albumin fusion proteins and/or polynucleotides ofthe invention are used in any combination with RIFABUTIN™,CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat orprevent an opportunistic Mycobacterium tuberculosis infection. Inanother specific embodiment, albumin fusion proteins and/orpolynucleotides of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat orprevent an opportunistic cytomegalovirus infection. In another specificembodiment, albumin fusion proteins and/or polynucleotides of theinvention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™,and/or KETOCONAZOLE™ to prophylactically treat or prevent anopportunistic fungal infection. In another specific embodiment, albuminfusion proteins and/or polynucleotides of the invention are used in anycombination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, albumin fusionproteins and/or polynucleotides of the invention are used in anycombination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylacticallytreat or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, albumin fusion proteins and/orpolynucleotides of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[0308] In a further embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantibiotic agent. Antibiotic agents that may be administered with thealbumin fusion proteins and/or polynucleotides of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

[0309] In other embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withimmunestimulants. Immunostimulants that may be administered incombination with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, levamisole (e.g.,ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g.interferon alpha), and interleukins (e.g., IL-2).

[0310] In other embodiments, albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withimmunosuppressive agents. Immunosuppressive agents that may beadministered in combination with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,steroids, cyclosporine, cyclosporine analogs, cyclophosphamidemethylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin,and other immunosuppressive agents that act by suppressing the functionof responding T cells. Other immunosuppressive agents that may beadministered in combination with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin,leflunomide, mizoribine (BREDININ), brequinar, deoxyspergalin, andazaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™,NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus),CELLCEPT® (mycophenolate motefil, of which the active metabolite ismycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids,adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™(prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™(methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

[0311] In an additional embodiment, albumin fusion proteins and/orpolynucleotides of the invention are administered alone or incombination with one or more intravenous immune globulin preparations.Intravenous immune globulin preparations that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™^(M),GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In aspecific embodiment, albumin fusion proteins and/or polynucleotides ofthe invention are administered in combination with intravenous immuneglobulin preparations in transplantation therapy (e.g., bone marrowtransplant).

[0312] In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered alone or incombination with an anti-inflammatory agent. Anti-inflammatory agentsthat may be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,corticosteroids (e.g. betamethasone, budesonide, cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs(e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, andtolmetin.), as well as antihistamines, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

[0313] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

[0314] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0315] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[0316] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[0317] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include, but are not limited to, platelet factor 4; protaminesulphate; sulphated chitin derivatives (prepared from queen crabshells), (Murata et al., Cancer Res. 51:22-26, (1991)); SulphatedPolysaccharide Peptidoglycan Complex (SP-PG) (the function of thiscompound may be enhanced by the presence of steroids such as estrogen,and tamoxifen citrate); Staurosporine; modulators of matrix metabolism,including for example, proline analogs, cishydroxyproline,d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone;Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum;ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992));Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992));Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin(Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate(“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987));anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute);Lobenzarit disodium (N-(2) -carboxyphenyl-4-chloroanthronilic aciddisodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992));and metalloproteinase inhibitors such as BB94.

[0318] Additional anti-angiogenic factors that may also be utilizedwithin the context of the present invention include Thalidomide,(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J.Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; P1-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

[0319] Anti-angiogenic agents that may be administed in combination withthe compounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositons of the invention include, but are not limited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositons of the inventioninclude, but are not Imited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.),and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectlyinhibit angiogenesis. Examples of indirect inhibitors of angiogenesiswhich may be administered in combination with the compositons of theinvention include, but are not limited to, IM-862 (Cytran, Kirkland,Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosanpolysulfate (Georgetown University, Washington, D.C.).

[0320] In particular embodiments, the use of compositions of theinvention in combination with anti-angiogenic agents is contemplated forthe treatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

[0321] In a particular embodiment, the use of compositions of theinvention in combination with anti-angiogenic agents is contemplated forthe treatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

[0322] In another embodiment, the polynucleotides encoding a polypeptideof the present invention are administered in combination with anangiogenic protein, or polynucleotides encoding an angiogenic protein.Examples of angiogenic proteins that may be administered with thecompositions of the invention include, but are not limited to, acidicand basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermalgrowth factor alpha and beta, platelet-derived endothelial cell growthfactor, platelet-derived growth factor, tumor necrosis factor alpha,hepatocyte growth factor, insulin-like growth factor, colony stimulatingfactor, macrophage colony stimulating factor, granulocyte/macrophagecolony stimulating factor, and nitric oxide synthase.

[0323] In additional embodiments, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the albumin fusionproteins and/or polynucleotides of the invention include, but are notlimited to alkylating agents such as nitrogen mustards (for example,Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide,Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines andmethylmelamines (for example, Hexamethylmelamine and Thiotepa), alkylsulfonates (for example, Busulfan), nitrosoureas (for example,Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), andStreptozocin (streptozotocin)), triazenes (for example, Dacarbazine(DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (forexample, Methotrexate (amethopterin)), pyrimidine analogs (for example,Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine;FudR), and Cytarabine (cytosine arabinoside)), purine analogs andrelated inhibitors (for example, Mercaptopurine (6-mercaptopurine;6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin(2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB,vinblastine sulfate)) and Vincristine (vincristine sulfate)),epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics(for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin;rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), andMitomycin (mitomycin C), enzymes (for example, L-Asparaginase),biological response modifiers (for example, Interferon-alpha andinterferon-alpha-2b), platinum coordination compounds (for example,Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone),substituted ureas (for example, Hydroxyurea), methylhydrazinederivatives (for example, Procarbazine (N-methylhydrazine; M1H),adrenocorticosteroids (for example, Prednisone), progestins (forexample, Hydroxyprogesterone caproate, Medroxyprogesterone,Medroxyprogesterone acetate, and Megestrol acetate), estrogens (forexample, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate,Estradiol, and Ethinyl estradiol), antiestrogens (for example,Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone),antiandrogens (for example, Flutamide), gonadotropin-releasing horomoneanalogs (for example, Leuprolide), other hormones and hormone analogs(for example, methyltestosterone, estramustine, estramustine phosphatesodium, chlorotrianisene, and testolactone), and others (for example,dicarbazine, glutamic acid, and mitotane).

[0324] In one embodiment, the compositions of the invention areadministered in combination with one or more of the following drugs:infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche,RO-32-3555), Leflunomide (also known as Arava™ from Hoechst MarionRoussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinrafrom Amgen, Inc.)

[0325] In a specific embodiment, compositions of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or combination of one or more of thecomponents of CHOP. In one embodiment, the compositions of the inventionare administered in combination with anti-CD20 antibodies, humanmonoclonal anti-CD20 antibodies. In another embodiment, the compositionsof the invention are administered in combination with anti-CD20antibodies and CHOP, or anti-CD20 antibodies and any combination of oneor more of the components of CHOP, particularly cyclophosphamide and/orprednisone. In a specific embodiment, compositions of the invention areadministered in combination with Rituximab. In a further embodiment,compositions of the invention are administered with Rituximab and CHOP,or Rituximab and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. In a specificembodiment, compositions of the invention are administered incombination with tositumomab. In a further embodiment, compositions ofthe invention are administered with tositumomab and CHOP, or tositumomaband any combination of one or more of the components of CHOP,particularly cyclophosphamide and/or prednisone. The anti-CD20antibodies may optionally be associated with radioisotopes, toxins orcytotoxic prodrugs.

[0326] In another specific embodiment, the compositions of the inventionare administered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radisotopes. Particularly preferred isotopesare ⁹⁰Y and ¹¹¹In.

[0327] In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withcytokines. Cytokines that may be administered with the albumin fusionproteins and/or polynucleotides of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, ILlS,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,albumin fusion proteins and/or polynucleotides of the invention may beadministered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, and IL-21.

[0328] In one embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withmembers of the TNF family. TNF, TNF-related or TNF-like molecules thatmay be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL. CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),OPG, and neutrokine-alpha (International Publication No. WO 98/18921,OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30,CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095),DR3 (International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TRANK, TR9 (International Publication No. WO 98/56892), TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153.

[0329] In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withangiogenic proteins. Angiogenic proteins that may be administered withthe albumin fusion proteins and/or polynucleotides of the inventioninclude but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are herein incorporated by reference in their entireties.

[0330] In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withFibroblast Growth Factors. Fibroblast Growth Factors that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10. FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0331] In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withhematopoietic growth factors. Hematopoietic growth factors that may beadministered with the albumin fusion proteins and/or polynucleotides ofthe invention include, but are not limited to, granulocyte macrophagecolony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™),granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™),macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin(epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand,steel factor), megakaryocyte colony stimulating factor, PIXY321 (aGMCSF/IL-3 fusion protein), interleukins, especially any one or more ofIL-1 through IL-12, interferon-gamma, or thrombopoietin. In certainembodiments, albumin fusion proteins and/or polynucleotides of thepresent invention are administered in combination with adrenergicblockers, such as, for example, acebutolol, atenolol, betaxolol,bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol,penbutolol, pindolol, propranolol, sotalol, and timolol.

[0332] In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination with anantiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis,digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide,lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetylprocainamide, propafenone, propranolol, quinidine, sotalol, tocainide,and verapamil).

[0333] In another embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withdiuretic agents, such as carbonic anhydrase-inhibiting agents (e.g.,acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics(e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibitNa⁺-K⁺-2Cl⁻ symport (e.g., furosemide, bumetamide, azosemide,piretamide, tripamide, ethacrynic acid, muzolimine, and torsemide),thiazide and thiazide-like diuretics (e.g., bendroflumethiazide,benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide,methyclothiazide, polythiazide, trichormethiazide, chlorthalidone,indapamide, metolazone, and quinethazone), potassium sparing diuretics(e.g., amiloride and triamterene), and mineralcorticoid receptorantagonists (e.g., spironolactone, canrenone, and potassium canrenoate).

[0334] In one embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withtreatments for endocrine and/or hormone imbalance disorders. Treatmentsfor endocrine and/or hormone imbalance disorders include, but are notlimited to, ¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³I;recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin);growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonistssuch as PARLODEL™ (bromocriptine); somatostatin analogs such asSANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™,A.P.L. and PROFASI™ (chorionic gonadotropin (CG)), PERGONAL™(menotropins), and MEFRODIN™ (urofollitropin (uFSH)); synthetic humangonadotropin releasing hormone preparations such as FACTREL™ andLUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonistssuch as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate),SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate);synthetic preparations of thyrotropin-releasing hormone such as RELEFACTTRH™ and THYPINONE™ (protirelin); recombinant human TSH such asTHYROGEN™; synthetic preparations of the sodium salts of the naturalisomers of thyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™(levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroinesodium), and THYROLAR™ (liotrix); antithyroid compounds such as6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazoleand TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole);beta-adrenergic receptor antagonists such as propranolol and esmolol;Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrastagents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodiumipodate).

[0335] Additional treatments for endocrine and/or hormone imbalancedisorders include, but are not limited to, estrogens or congugatedestrogens such as ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol),PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone),ESTROV IS (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ andVALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECTLA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen),SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™(hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™(medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™(megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ andAYGESTIN™ (norethindrone acetate); progesterone implants such asNORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins suchas RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™(norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device thatreleases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™,NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinylestradiol/norethindrone), LEVLEN™, NORDETTE™, TR1-LEVLEN™ andTRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™(ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodioldiacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™(norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinylestradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinylestradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), andOVRETTE™ (norgestrel).

[0336] Additional treatments for endocrine and/or hormone imbalancedisorders include, but are not limited to, testosterone esters such asmethenolone acetate and testosterone undecanoate; parenteral and oralandrogens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosteronepropionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™(testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™(fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™(methyltestosterone), and OXANDRIN™ (oxandrolone); testosteronetransdermal systems such as TESTODERM; androgen receptor antagonist and5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate),EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropichormone preparations such as CORTROSYN™ (cosyntropin); adrenocorticalsteroids and their synthetic analogs such as ACLOVATE™ (alclometasonedipropionate), CYCLOCORT™ (amcinonide), BECLOVENT™ and VANCERIL™(beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ andUTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasonedipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate),CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate),BETA-VAL™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasolpropionate), CLODERM™ (clocortolone pivalate), CORTEF™ and HYDROCORTONE™(cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol(hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate),HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate),A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodiumsuccinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONEACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide),TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™(dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™(dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasonediacetate), FLORINEF ACEFATE™ (fludrocortisone acetate), AEROBID™ andNASALIDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide),LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™(flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone),MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™(methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™(methylprednisolone sodium succinate), ELOCON™ (mometasone furoate),HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone),ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodiumphosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™(prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™(triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™(triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide);inhibitors of biosynthesis and action of adrenocortical steroids such asCYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™(trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or humaninsulin or mixtures thereof; insulin analogs; recombinant human insulinsuch as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ andTOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide,MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide),and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone,pioglitazone, and alpha-glucosidase inhibitors; bovine or porcineglucagon; somatostatins such as SANDOSTATIN™ (octreotide); anddiazoxides such as PROGLYCEM™ (diazoxide).

[0337] In one embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withtreatments for uterine motility disorders. Treatments for uterinemotility disorders include, but are not limited to, estrogen drugs suchas conjugated estrogens (e.g., PREMARIN® and ESTRATAB®), estradiols(e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene;progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR®(norethidrone acetate), PROMETRIUM® progesterone, and megestrolacetate); and estrogen/progesterone combination therapies such as, forexample, conjugated estrogens/medroxyprogesterone (e.g., PREMPRO™ andPREMPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g.,FEMHRT™).

[0338] In an additional embodiment, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withdrugs effective in treating iron deficiency and hypochromic anemias,including but not limited to, ferrous sulfate (iron sulfate. FEOSOL™),ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g., FERGON™),polysaccharide-iron complex (e.g., NIFEREX™), iron dextran injection(e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂,cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™),hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid,5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt ofleucovorin), transferrin or ferritin.

[0339] In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withagents used to treat psychiatric disorders. Psychiatric drugs that maybe administered with the albumin fusion proteins and/or polynucleotidesof the invention include, but are not limited to, antipsychotic agents(e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine,haloperidol, loxapine, mesoridazine, molindone, olanzapine,perphenazine, pimozide, quetiapine, risperidone, thioridazine,thiothixene, trifluoperazine, and triflupromazine), antimanic agents(e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithiumcitrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion,citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine,imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone,nortriptyline, paroxetine, phenelzine, protriptyline, sertraline,tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxietyagents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate,diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants(e.g., d-amphetamine, methylphenidate, and pemoline).

[0340] In other embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withagents used to treat neurological disorders. Neurological agents thatmay be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide,phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium,felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine,tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam),antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline,amantidine, bromocriptine, pergolide, ropinirole, pramipexole,benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl,tolcapone), and ALS therapeutics (e.g. riluzole).

[0341] In another embodiment, albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withvasodilating agents and/or calcium channel blocking agents. Vasodilatingagents that may be administered with the albumin fusion proteins and/orpolynucleotides of the invention include, but are not limited to,Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine,isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat,fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril,spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbidedinitrate, isosorbide mononitrate, and nitroglycerin). Examples ofcalcium channel blocking agents that may be administered in combinationwith the albumin fusion proteins and/or polynucleotides of the inventioninclude, but are not limited to amlodipine, bepridil, diltiazem,felodipine, flunarizine, isradipine, nicardipine, nifedipine,nimodipine, and verapamil.

[0342] In certain embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withtreatments for gastrointestinal disorders. Treatments forgastrointestinal disorders that may be administered with the albuminfusion protein and/or polynucleotide of the invention include, but arenot limited to, H2 histamine receptor antagonists (e.g., TAGAMET™(cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™(nizatidine)); inhibitors of H⁺, K⁺ ATPase (e.g., PREVACID™(lansoprazole) and PRILOSECM (omeprazole)); Bismuth compounds (e.g.,PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate));various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™(misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g.,surfactant laxatives, stimulant laxatives, saline and osmoticlaxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate),MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)),synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide),antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetronhydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine,perphenazine, prochlorperazine, promethazine, thiethylperazine,triflupromazine, domperidone, haloperidol, droperidol,trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, andnabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide andchlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholicacid; and pancreatic enzyme preparations such as pancreatin andpancrelipase.

[0343] In additional embodiments, the albumin fusion proteins and/orpolynucleotides of the invention are administered in combination withother therapeutic or prophylactic regimens, such as, for example,radiation therapy.

[0344] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions comprising albumin fusionproteins of the invention. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

[0345] Gene Therapy

[0346] Constructs encoding albumin fusion proteins of the invention canbe used as a part of a gene therapy protocol to deliver therapeuticallyeffective doses of the albumin fusion protein. A preferred approach forin vivo introduction of nucleic acid into a cell is by use of a viralvector containing nucleic acid, encoding an albumin fusion protein ofthe invention. Infection of cells with a viral vector has the advantagethat a large proportion of the targeted cells can receive the nucleicacid. Additionally, molecules encoded within the viral vector, e.g., bya cDNA contained in the viral vector, are expressed efficiently in cellswhich have taken up viral vector nucleic acid.

[0347] Retrovirus vectors and adeno-associated virus vectors can be usedas a recombinant gene delivery system for the transfer of exogenousnucleic acid molecules encoding albumin fusion proteins in vivo. Thesevectors provide efficient delivery of nucleic acids into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. The development of specialized cell lines (termed“packaging cells”) which produce only replication-defective retroviruseshas increased the utility of retroviruses for gene therapy, anddefective retroviruses are characterized for use in gene transfer forgene therapy purposes (for a review see Miller, A. D. (1990) Blood76:271). A replication defective retrovirus can be packaged into virionswhich can be used to infect a target cell through the use of a helpervirus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al., (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14 and other standard laboratory manuals.

[0348] Another viral gene delivery system useful in the presentinvention uses adenovirus-derived vectors. The genome of an adenoviruscan be manipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al.,BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434(1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 dl324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells and can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al., (1992) citedsupra). Furthermore, the virus particle is relatively stable andamenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al., cited supra;Haj-Ahmand et al., J. Virol. 57:267 (1986)).

[0349] In another embodiment, non-viral gene delivery systems of thepresent invention rely on endocytic pathways for the uptake of thesubject nucleotide molecule by the targeted cell. Exemplary genedelivery systems of this type include liposomal derived systems,poly-lysine conjugates and artificial viral envelopes. In arepresentative embodiment, a nucleic acid molecule encoding an albuminfusion protein of the invention can be entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins) and (optionally)which are tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCTpublication WO91/06309; Japanese patent application 1047381; andEuropean patent publication EP-A43075).

[0350] Gene delivery systems for a gene encoding an albumin fusionprotein of the invention can be introduced into a patient by any of anumber of methods. For instance, a pharmaceutical preparation of thegene delivery system can be introduced systemically, e.g. by intravenousinjection, and specific transduction of the protein in the target cellsoccurs predominantly from specificity of transfection provided by thegene delivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the recombinant gene is more limited with introduction intothe animal being quite localized. For example, the gene delivery vehiclecan be introduced by catheter (see U.S. Pat. No. 5,328,470) or byStereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057). Thepharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Where the albumin fusion protein can be produced intact fromrecombinant cells, e.g. retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the albuminfusion protein.

[0351] Additional Gene Therapy Methods

[0352] Also encompassed by the invention are gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofan albumin fusion protein of the invention. This method requires apolynucleotide which codes for an albumin fusion protein of the presentinvention operatively linked to a promoter and any other geneticelements necessary for the expression of the fusion protein by thetarget tissue. Such gene therapy and delivery techniques are known inthe art, see, for example, WO90/11092, which is herein incorporated byreference.

[0353] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide encoding an albumin fusion protein of the presentinvention ex vivo, with the engineered cells then being provided to apatient to be treated with the fusion protein of the present invention.Such methods are well-known in the art. For example, see Belldegrun, A.,et al., J. Natl Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al.,Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60:221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato,L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al.,Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated byreference. In one embodiment, the cells which are engineered arearterial cells. The arterial cells may be reintroduced into the patientthrough direct injection to the artery, the tissues surrounding theartery, or through catheter injection.

[0354] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0355] In one embodiment, polynucleotides encoding the albumin fusionproteins of the present invention is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, polynucleotides encoding the albuminfusion proteins of the present invention can also be delivered inliposome formulations and lipofectin formulations and the like can beprepared by methods well known to those skilled in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0356] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0357] Any strong promoter known to those skilled in the art can be usedfor driving the expression of the polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the NMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for the gene corresponding to the Therapeuticprotein portion of the albumin fusion proteins of the invention.

[0358] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0359] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0360] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0361] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0362] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0363] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0364] In certain embodiments, the polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

[0365] Cationic liposomes are readily available. Forexample, >N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA)liposomes are particularly useful and are available under the trademarkLipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner etal., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is hereinincorporated by reference). Other commercially available liposomesinclude transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0366] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0367] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0368] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0369] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mNM Tris/NaCl,sonicated, and then the preformed liposomes are mixed directly with theDNA. The liposome and DNA form a very stable complex due to binding ofthe positively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys.Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978);Schaefer-Ridder et al., Science 215:166 (1982)), which are hereinincorporated by reference.

[0370] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0371] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication no. WO94/9469 provide methods for delivering DNA-cationic lipid complexes tomammals.

[0372] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding an albumin fusion protein of the present invention.Retroviruses from which the retroviral plasmid vectors may be derivedinclude, but are not limited to, Moloney Murine Leukemia Virus, spleennecrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosisvirus, gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

[0373] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0374] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding an albumin fusionprotein of the present invention. Such retroviral vector particles thenmay be employed, to transduce eukaryotic cells, either in vitro or invivo. The transduced eukaryotic cells will express a fusion protin ofthe present invention.

[0375] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses fusion protein ofthe present invention, and at the same time is inactivated in terms ofits ability to replicate in a normal lytic viral life cycle. Adenovirusexpression is achieved without integration of the viral DNA into thehost cell chromosome, thereby alleviating concerns about insertionalmutagenesis. Furthermore, adenoviruses have been used as live entericvaccines for many years with an excellent safety profile (Schwartz etal. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirusmediated gene transfer has been demonstrated in a number of instancesincluding transfer of alpha-1-antitrypsin and CFTR to the lungs ofcotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431434;Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.Acad. Sci. USA 76:6606).

[0376] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0377] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0378] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0379] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructis inserted into the AAV vector using standard cloning methods, such asthose found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989). The recombinant AAV vector is thentransfected into packaging cells which are infected with a helper virus,using any standard technique, including lipofection, electroporation,calcium phosphate precipitation, etc. Appropriate helper viruses includeadenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.Once the packaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct. These viral particles are then used to transduce eukaryoticcells, either ex vivo or in vivo. The transduced cells will contain thepolynucleotide construct integrated into its genome, and will express afsuion protein of the invention.

[0380] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding a polypeptide of the present invention) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), which are hereinencorporated by reference. This method involves the activation of a genewhich is present in the target cells, but which is not normallyexpressed in the cells, or is expressed at a lower level than desired.

[0381] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[0382] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0383] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0384] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[0385] The polynucleotide encoding an albumin fusion protein of thepresent invention may contain a secretory signal sequence thatfacilitates secretion of the protein. Typically, the signal sequence ispositioned in the coding region of the polynucleotide to be expressedtowards or at the 5′ end of the coding region. The signal sequence maybe homologous or heterologous to the polynucleotide of interest and maybe homologous or heterologous to the cells to be transfected.Additionally, the signal sequence may be chemically synthesized usingmethods known in the art.

[0386] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers (Kaneda et al., Science 243:375 (1989)).

[0387] A preferred method of local administration is by directinjection. Preferably, an albumin fusion protein of the presentinvention complexed with a delivery vehicle is administered by directinjection into or locally within the area of arteries. Administration ofa composition locally within the area of arteries refers to injectingthe composition centimeters and preferably, millimeters within arteries.

[0388] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0389] Therapeutic compositions useful in systemic administration,include fusion proteins of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site. In specificembodiments, suitable delivery vehicles for use with systemicadministration comprise liposomes comprising albumin fusion proteins ofthe invention for targeting the vehicle to a particular site.

[0390] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0391] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.

[0392] Albumin fusion proteins of the present invention can beadministered to any animal, preferably to mammals and birds. Preferredmammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle,horses and pigs, with humans being particularly preferred.

[0393] Biological Activities

[0394] Albumin fusion proteins and/or polynucleotides encoding albuminfusion proteins of the present invention, can be used in assays to testfor one or more biological activities. If an albumin fusion proteinand/or polynucleotide exhibits an activity in a particular assay, it islikely that the Therapeutic protein corresponding to the fusion porteinmay be involved in the diseases associated with the biological activity.Thus, the fusion protein could be used to treat the associated disease.

[0395] Members of the secreted family of proteins are believed to beinvolved in biological activities associated with, for example, cellularsignaling. Accordingly, albumin fusion proteins of the invention andpolynucleotides encoding these protiens, may be used in diagnosis,prognosis, prevention and/or treatment of diseases and/or disordersassociated with aberrant activity of secreted polypeptides.

[0396] In a preferred embodiment, albumin fusion proteins of theinvention comprising a Therapeutic protein portion corresponding to EPO,immunoglobulins, hirudin, applaggin, serum cholinesterase, alpha-iantitrypsin, aprotinin, and coagulation factors in both pre and activeforms (e.g., including, but not limited to, von Willebrand factor,fibrinogen, factor II, factor VII, factor VIIA activated factor, factorVIII, factor IX, factor X, factor XIII, cl inactivator, antithrombinIII, thrombin and prothrombin, apo-lipoprotein, c-reactive protein, andprotein C) and/or fragments and/or variants thereof may be used tomodulate hemostatic (the stopping of bleeding) or thrombolytic (clotdissolving) activity and/or treat, prevent, diagnose, prognose, and/ordetect blood-related disorders or cardiovascular disorders and/ordiseases, disorders or conditions as described under “Blood RelatedDisorders”, “Anti-Angiogenesis Activity”, and/or “CardiovascularDisorders” infra.

[0397] In a preferred embodiment, albumin fusion proteins of theinvention comprising a Therapeutic protein portion corresponding toInterferon alpha, G-CSF, GM-CSF, scatter factor, MCP/MCAF, M-CSF and/orfragments and/or variants thereof may be used to treat, prevent,diagnose, prognose, and/or detect diseases or disorders of the immunesystem, or diseases, disorders or conditions as described under “ImmuneActivity”, “Infectious Disease”, and/or “Hyperproliferative Disorders”infra.

[0398] In a preferred embodiment, albumin fusion proteins of theinvention comprising a Therapeutic protein portion corresponding tohuman Growth hormone and/or fragments and/or variants thereof may beused to treat, prevent, diagnose, prognose, and/or detect disease,disorders and/or conditions related to growth hormone deficiency,including but not limited to, Acromegaly, Growth failure, Growth failureand endogenous growth hormone replacement, Growth hormone deficiency,Growth failure and growth retardation, Prader-Willi syndrome in children2 years or older, Growth deficiencies, Postmenopausal osteoporosis,burns, cachexia, cancer cachexia, dwarfism, metabolic disorders,obesity, renal failure, Turner's Syndrome, fibromyalgia, fracturetreatment, frailty, or as described under “Endocrine Disorders”, “WoundHealing and Epithelial Cell Proliferation”, and/or “HyperproliferativeDisorders” infra.

[0399] In preferred embodiments, fusion proteins of the presentinvention may be used in the diagnosis, prognosis, prevention and/ortreatment of diseases and/or disorders relating to diseases anddisorders of the endocrine system (see, for example, “EndocrineDisorders” section below), the nervous system (see, for example,“Neurological Disorders” section below), the immune system (see, forexample, “Immune Activity” section below), respiratory system (see, forexample, “Respiratory Disorders” section below), cardiovascular system(see, for example, “Cardiovascular Disorders” section below),reproductive system (see, for example, “Reproductive System Disorders”section below) digestive system (see, for example, “GastrointestinalDisorders” section below), diseases and/or disorders relating to cellproliferation (see, for example, “Hyperproliferative Disorders” sectionbelow), and/or diseases or disorders relating to the blood (see, forexample, “Blood-Related Disorders” section below).

[0400] In preferred embodiments, the present invention encompasses amethod of treating a disease or disorder listed in the “PreferredIndication Y” column of Table 1 comprising administering to a patient inwhich such treatment, prevention or amelioration is desired an albuminfusion protein of the invention that comprises a Therapeutic proteinportion corresponding to a Therapeutic protein disclosed in the“Therapeutic Protein X” column of Table 1 (in the same row as thedisease or disorder to be treated is listed in the “Preferred IndicationY” column of Table 1) in an amount effective to treat, prevent orameliorate the disease or disorder.

[0401] In certain embodiments, an albumin fusion protein of the presentinvention may be used to diagnose and/or prognose diseases and/ordisorders associated with the tissue(s) in which the gene correspondingto the Therapeutic protein portion of the fusion portien of theinvention is expressed.

[0402] Thus, fusion proteins of the invention and polynucleotidesencoding albumin fusion proteins of the invention are useful in thediagnosis, detection and/or treatment of diseases and/or disordersassociated with activities that include, but are not limited to,prohormone activation, neurotransmitter activity, cellular signaling,cellular proliferation, cellular differentiation, and cell migration.

[0403] More generally, fusion proteins of the invention andpolynucleotides encoding albumin fusion proteins of the invention may beuseful for the diagnosis, prognosis, prevention and/or treatment ofdiseases and/or disorders associated with the following systems.

[0404] Immune Activity

[0405] Albumin fusion proteins of the invention and polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, diagnosing and/or prognosing diseases, disorders,and/or conditions of the immune system, by, for example, activating orinhibiting the proliferation, differentiation, or mobilization(chemotaxis) of immune cells. Immune cells develop through a processcalled hematopoiesis, producing myeloid (platelets, red blood cells,neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cellsfrom pluripotent stem cells. The etiology of these immune diseases,disorders, and/or conditions may be genetic, somatic, such as cancer andsome autoimmune diseases, acquired (e.g., by chemotherapy or toxins), orinfectious. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as a marker or detector of a particular immune system disease ordisorder.

[0406] In another embodiment, a fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention, maybe used to treat diseases and disorders of the immune system and/or toinhibit or enhance an immune response generated by cells associated withthe tissue(s) in which the polypeptide of the invention is expressed.

[0407] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, diagnosing, and/or prognosing immunodeficiencies,including both congenital and acquired immunodeficiencies. Examples of Bcell immunodeficiencies in which immunoglobulin levels B cell functionand/or B cell numbers are decreased include: X-linked agammaglobulinemia(Bruton's disease), X-linked infantile agammaglobulinemia, X-linkedimmunodeficiency with hyper IgM, non X-linked immunodeficiency withhyper IgM. X-linked lymphoproliferative syndrome (XLP),agammaglobulinemia including congenital and acquired agammaglobulinemia,adult onset agammaglobulinemia, late-onset agammaglobulinemia,dysgammaglobulinemia, hypogammaglobulinemia, unspecifiedhypogammaglobulinemia, recessive agammaglobulinemia (Swiss type),Selective IgM deficiency, selective IgA deficiency, selective IgGsubclass deficiencies, IgG subclass deficiency (with or without IgAdeficiency), Ig deficiency with increased IgM, IgG and IgA deficiencywith increased IgM, antibody deficiency with normal or elevated Igs, Igheavy chain deletions, kappa chain deficiency, B celllymphoproliferative disorder (BLPD), common variable immunodeficiency(CVID), common variable immunodeficiency (CVI) (acquired), and transienthypogammaglobulinemia of infancy.

[0408] In specific embodiments, ataxia-telangiectasia or conditionsassociated with ataxia-telangiectasia are treated, prevented, diagnosed,and/or prognosing using the, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention.

[0409] Examples of congenital immunodeficiencies in which T cell and/orB cell function and/or number is decreased include, but are not limitedto: DiGeorge anomaly, severe combined immunodeficiencies (SCID)(including, but not limited to, X-linked SCID, autosomal recessive SCID,adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP)deficiency, Class II MHC deficiency (Bare lymphocyte syndrome),Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia,third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronicmucocutaneous candidiasis, natural killer cell deficiency (NK),idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant Tcell defect (unspecified), and unspecified immunodeficiency of cellmediated immunity.

[0410] In specific embodiments, DiGeorge anomaly or conditionsassociated with DiGeorge anomaly are treated, prevented, diagnosed,and/or prognosed using fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention.

[0411] Other immunodeficiencies that may be treated, prevented,diagnosed, and/or prognosed using fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, include, but are not limited to, chronic granulomatousdisease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyteglucose-6-phosphate dehydrogenase deficiency, X-linkedlymphoproliferative syndrome (XLP), leukocyte adhesion deficiency,complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7,C8 and/or C9 deficiencies), reticular dysgenesis, thymicalymphoplasia-aplasia, immunodeficiency with thymoma, severe congenitalleukopenia, dysplasia with immunodeficiency, neonatal neutropenia, shortlimbed dwarfism, and Nezelof syndrome-combined immunodeficiency withIgs.

[0412] In a preferred embodiment, the immunodeficiencies and/orconditions associated with the immunodeficiencies recited above aretreated, prevented, diagnosed and/or prognosed using fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention.

[0413] In a preferred embodiment fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used as an agent to boost immunoresponsiveness among immunodeficientindividuals. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioncould be used as an agent to boost immunoresponsiveness among B celland/or T cell immunodeficient individuals.

[0414] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in treating, preventing, diagnosing and/or prognosing autoimmunedisorders. Many autoimmune disorders result from inappropriaterecognition of self as foreign material by immune cells. Thisinappropriate recognition results in an immune response leading to thedestruction of the host tissue. Therefore, the administration of fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention that can inhibit an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in preventing autoimmune disorders.

[0415] Autoimmune diseases or disorders that may be treated, prevented,diagnosed and/or prognosed by fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, one or more of the following: systemiclupus erythematosus, rheumatoid arthritis, ankylosing spondylitis,multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis,autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia,autoimmune thrombocytopenia purpura, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g.,Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome,Pemphigus vulgaris, myasthenia gravis, Grave's disease(hyperthyroidism), and insulin-resistant diabetes mellitus.

[0416] Additional disorders that are likely to have an autoimmunecomponent that may be treated, prevented, and/or diagnosed with thealbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, but are not limitedto, type II collagen-induced arthritis, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia,polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmunepulmonary inflammation, autism, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disorders.

[0417] Additional disorders that are likely to have an autoimmunecomponent that may be treated, prevented, diagnosed and/or prognosedwith the albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention include, but are notlimited to, scleroderma with anti-collagen antibodies (oftencharacterized, e.g., by nucleolar and other nuclear antibodies), mixedconnective tissue disease (often characterized, e.g., by antibodies toextractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis(often characterized, e.g., by nonhistone ANA), pernicious anemia (oftencharacterized, e.g., by antiparietal cell, microsomes, and intrinsicfactor antibodies), idiopathic Addison's disease (often characterized,e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, e.g., by antispermatozoal antibodies),glomerulonephritis (often characterized, e.g., by glomerular basementmembrane antibodies or immune complexes), bullous pemphigoid (oftencharacterized, e.g., by IgG and complement in basement membrane),Sjogren's syndrome (often characterized, e.g., by multiple tissueantibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus(often characterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

[0418] Additional disorders that may have an autoimmune component thatmay be treated, prevented, diagnosed and/or prognosed with the albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention include, but are not limited to,chronic active hepatitis (often characterized, e.g., by smooth muscleantibodies), primary biliary cirrhosis (often characterized, e.g., bymitochondria antibodies), other endocrine gland failure (oftencharacterized, e.g., by specific tissue antibodies in some cases),vitiligo (often characterized, e.g., by melanocyte antibodies),vasculitis (often characterized, e.g., by Ig and complement in vesselwalls and/or low serum complement), post-MI (often characterized, e.g.,by myocardial antibodies), cardiotomy syndrome (often characterized,e.g., by myocardial antibodies), urticaria (often characterized, e.g.,by IgG and IgM antibodies to IgE), atopic dermatitis (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), asthma (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), and many otherinflammatory, granulomatous, degenerative, and atrophic disorders.

[0419] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, diagnosed and/or prognosed using forexample, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention. In a specificpreferred embodiment, rheumatoid arthritis is treated, prevented, and/ordiagnosed using fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention.

[0420] In another specific preferred embodiment, systemic lupuserythematosus is treated, prevented, and/or diagnosed using fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention. In another specific preferred embodiment,idiopathic thrombocytopenia purpura is treated, prevented, and/ordiagnosed using fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention.

[0421] In another specific preferred embodiment IgA nephropathy istreated, prevented, and/or diagnosed using fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention.

[0422] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, diagnosed and/or prognosed using fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention.

[0423] In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused as a immunosuppressive agent(s).

[0424] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, prognosing, and/or diagnosing diseases, disorders,and/or conditions of hematopoietic cells. Albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention could be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with a decrease in certain (or many) types hematopoieticcells, including but not limited to, leukopenia, neutropenia, anemia,and thrombocytopenia. Alternatively, fusion proteins of the inventionand/or polynucleofides encoding albumin fusion proteins of the inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with an increase in certain (or many) types of hematopoieticcells, including but not limited to, histiocytosis.

[0425] Allergic reactions and conditions, such as asthma (particularlyallergic asthma) or other respiratory problems, may also be treated,prevented, diagnosed and/or prognosed using fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention. Moreover, these molecules can be used to treat, prevent,prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenicmolecule, or blood group incompatibility.

[0426] Additionally, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe used to treat, prevent, diagnose and/or prognose IgE-mediatedallergic reactions. Such allergic reactions include, but are not limitedto, asthma, rhinitis, and eczema. In specific embodiments, fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention may be used to modulate IgE concentrations invitro or in vivo.

[0427] Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention have uses in thediagnosis, prognosis, prevention, and/or treatment of inflammatoryconditions. For example, since fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayinhibit the activation, proliferation and/or differentiation of cellsinvolved in an inflammatory response, these molecules can be used toprevent and/or treat chronic and acute inflammatory conditions. Suchinflammatory conditions include, but are not limited to, for example,inflammation associated with infection (e.g., septic shock, sepsis, orsystemic inflammatory response syndrome), ischemia-reperfusion injury,endotoxin lethality, complement-mediated hyperacute rejection,nephritis, cytokine or chemokine induced lung injury, inflammatory boweldisease, Crohn's disease, over production of cytokines (e.g., TNF orIL-1.), respiratory disorders (e.g., asthma and allergy);gastrointestinal disorders (e.g., inflammatory bowel disease); cancers(e.g., gastric, ovarian, lung, bladder, liver, and breast); CNSdisorders (e.g., multiple sclerosis; ischemic brain injury and/orstroke, traumatic brain injury, neurodegenerative disorders (e.g.,Parkinson's disease and Alzheimer's disease); AIDS-related dementia; andprion disease); cardiovascular disorders (e.g., atherosclerosis,myocarditis, cardiovascular disease, and cardiopulmonary bypasscomplications); as well as many additional diseases, conditions, anddisorders that are characterized by inflammation (e.g., hepatitis,rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis,dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemiclupus erythematosus, diabetes mellitus, and allogenic transplantrejection).

[0428] Because inflammation is a fundamental defense mechanism,inflammatory disorders can effect virtually any tissue of the body.Accordingly, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, have uses in thetreatment of tissue-specific inflammatory disorders, including, but notlimited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis,balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis,cervicitis, cholecystitis, chorditis, cochlitis, colitis,conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis,endocarditis, esophagitis, eustachitis, fibrositis, folliculitis,gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis,keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, mediaotitis, meningitis, metritis, mucitis, myocarditis, myosititis,myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis,pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis,scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis,steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis,urethritis, and vaginitis.

[0429] In specific embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, areuseful to diagnose, prognose, prevent, and/or treat organ transplantrejections and graft-versus-host disease. Organ rejection occurs by hostimmune cell destruction of the transplanted tissue through an immuneresponse. Similarly, an immune response is also involved in GVHD, but,in this case, the foreign transplanted immune cells destroy the hosttissues. Polypeptides, antibodies, or polynucleotides of the invention,and/or agonists or antagonists thereof, that inhibit an immune response,particularly the activation, proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD. In specific embodiments, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing experimental allergic and hyperacutexenograft rejection.

[0430] In other embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, areuseful to diagnose, prognose, prevent, and/or treat immune complexdiseases, including, but not limited to, serum sickness, poststreptococcal glomerulonephritis, polyarteritis nodosa, and immunecomplex-induced vasculitis.

[0431] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention can be used to treat,detect, and/or prevent infectious agents. For example, by increasing theimmune response, particularly increasing the proliferation activationand/or differentiation of B and/or T cells, infectious diseases may betreated, detected, and/or prevented. The immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also directly inhibit the infectious agent (refer tosection of application listing infectious agents, etc), withoutnecessarily eliciting an immune response.

[0432] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used as a vaccine adjuvant that enhances immune responsiveness to anantigen. In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an adjuvant to enhance tumor-specific immuneresponses.

[0433] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an adjuvant to enhance anti-viral immuneresponses. Anti-viral immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include virus and virusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions of the invention areused as an adjuvant to enhance an immune response to a virus, disease,or symptom selected from the group consisting of: AIDS, meningitis,Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specificembodiment, the compositions of the invention are used as an adjuvant toenhance an immune response to a virus, disease, or symptom selected fromthe group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue,rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza,measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever,herpes simplex, and yellow fever.

[0434] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an adjuvant to enhance anti-bacterial oranti-fungal immune responses. Anti-bacterial or anti-fungal immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include bacteria or fungus and bacteria or fungusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions of the invention areused as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:tetanus, Diphtheria, botulism, and meningitis type B.

[0435] In another specific embodiment, the compositions of the inventionare used as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group Bstreptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, and Borrelia burgdorferi.

[0436] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an adjuvant to enhance anti-parasitic immuneresponses. Anti-parasitic immune responses that may be enhanced usingthe compositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria) or Leishmania.

[0437] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also be employed to treat infectious diseases includingsilicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example,by preventing the recruitment and activation of mononuclear phagocytes.

[0438] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an antigen for the generation of antibodies toinhibit or enhance immune mediated responses against polypeptides of theinvention.

[0439] In one embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare administered to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody productionand immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE),and/or to increase an immune response.

[0440] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a stimulator of B cell responsiveness topathogens.

[0441] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an activator of T cells.

[0442] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent that elevates the immune status of anindividual prior to their receipt of immunosuppressive therapies.

[0443] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to induce higher affinity antibodies.

[0444] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to increase serum immunoglobulinconcentrations.

[0445] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to accelerate recovery ofimmunocompromised individuals.

[0446] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to boost immunoresponsiveness among agedpopulations and/or neonates.

[0447] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an immune system enhancer prior to, during, orafter bone marrow transplant and/or other transplants (e.g., allogeneicor xenogeneic organ transplantation). With respect to transplantation,compositions of the invention may be administered prior to, concomitantwith, and/or after transplantation. In a specific embodiment,compositions of the invention are administered after transplantation,prior to the beginning of recovery of T-cell populations. In anotherspecific embodiment, compositions of the invention are firstadministered after transplantation after the beginning of recovery of Tcell populations, but prior to full recovery of B cell populations.

[0448] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to boost immunoresponsiveness amongindividuals having an acquired loss of B cell function. Conditionsresulting in an acquired loss of B cell function that may be amelioratedor treated by administering the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, include, but are not limited to, HIV Infection, AIDS, bonemarrow transplant, and B cell chronic lymphocytic leukemia (CLL).

[0449] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to boost immunoresponsiveness amongindividuals having a temporary immune deficiency. Conditions resultingin a temporary immune deficiency that may be ameliorated or treated byadministering the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, and recovery fromsurgery.

[0450] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a regulator of antigen presentation by monocytes,dendritic cells, and/or B-cells. In one embodiment, albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention enhance antigen presentation or antagonizesantigen presentation in vitro or in vivo. Moreover, in relatedembodiments, this enhancement or antagonism of antigen presentation maybe useful as an anti-tumor treatment or to modulate the immune system.

[0451] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as an agent to direct an individual's immune systemtowards development of a humoral response (i.e. TH2) as opposed to a THIcellular response.

[0452] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a means to induce tumor proliferation and thusmake it more susceptible to anti-neoplastic agents. For example,multiple myeloma is a slowly dividing disease and is thus refractory tovirtually all anti-neoplastic regimens. If these cells were forced toproliferate more rapidly their susceptibility profile would likelychange.

[0453] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a stimulator of B cell production in pathologiessuch as AIDS, chronic lymphocyte disorder and/or Common VariableImmunodificiency.

[0454] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a therapy for generation and/or regeneration oflymphoid tissues following surgery, trauma or genetic defect. In anotherspecific embodiment, albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused in the pretreatment of bone marrow samples prior to transplant.

[0455] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a gene-based therapy for genetically inheriteddisorders resulting in immuno-incompetence/immunodeficiency such asobserved among SCID patients.

[0456] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a means of activating monocytes/macrophages todefend against parasitic diseases that effect monocytes such asLeishmania.

[0457] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a means of regulating secreted cytokines that areelicited by polypeptides of the invention.

[0458] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used in one or more of the applications decribed herein, as they mayapply to veterinary medicine.

[0459] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a means of blocking various aspects of immuneresponses to foreign agents or self. Examples of diseases or conditionsin which blocking of certain aspects of immune responses may be desiredinclude autoimmune disorders such as lupus, and arthritis, as well asimmunoresponsiveness to skin allergies, inflammation, bowel disease,injury and diseases/disorders associated with pathogens.

[0460] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a therapy for preventing the B cell proliferationand Ig secretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythematosus and multiplesclerosis.

[0461] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention invention are used as a inhibitor of B and/orT cell migration in endothelial cells. This activity disrupts tissuearchitecture or cognate responses and is useful, for example indisrupting immune responses, and blocking sepsis.

[0462] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a therapy for chronic hypergammaglobulinemiaevident in such diseases as monoclonal gammopathy of undeterminedsignificance (MGUS), Waldenstrom's disease, related idiopathicmonoclonal gammopathies, and plasmacytomas.

[0463] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be employed for instance to inhibit polypeptide chemotaxisand activation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain autoimmuneand chronic inflammatory and infective diseases. Examples of autoimmunediseases are described herein and include multiple sclerosis, andinsulin-dependent diabetes.

[0464] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayalso be employed to treat idiopathic hyper-eosinophilic syndrome by, forexample, preventing eosinophil production and migration.

[0465] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to enhance or inhibit complement mediated cell lysis.

[0466] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to enhance or inhibit antibody dependent cellularcytotoxicity.

[0467] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also be employed for treating atherosclerosis, forexample, by preventing monocyte infiltration in the artery wall.

[0468] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be employed to treat adult respiratory distress syndrome(ARDS).

[0469] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful for stimulating wound and tissue repair,stimulating angiogenesis, and/or stimulating the repair of vascular orlymphatic diseases or disorders. Additionally, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be used to stimulate the regeneration of mucosal surfaces.

[0470] In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to diagnose, prognose, treat, and/or prevent adisorder characterized by primary or acquired immunodeficiency,deficient serum immunoglobulin production, recurrent infections, and/orimmune system dysfunction. Moreover, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used to treat or prevent infections of the joints, bones, skin,and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g.,those disclosed herein), inflammatory disorders, and malignancies,and/or any disease or disorder or condition associated with theseinfections, diseases, disorders and/or malignancies) including, but notlimited to, CVID, other primary immune deficiencies, HIV disease, CLL,recurrent bronchitis, sinusitis, otitis media, conjunctivitis,pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpeszoster), and/or pneumocystis carnii. Other diseases and disorders thatmay be prevented, diagnosed, prognosed, and/or treated with fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention include, but are not limited to, HIVinfection, HTLV-BLV infection, lymphopenia, phagocyte bactericidaldysfunction anemia, thrombocytopenia, and hemoglobinuria.

[0471] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to treat, and/or diagnose an individual having common variableimmunodeficiency disease (“CVID”; also known as “acquiredagammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset ofthis disease.

[0472] In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be used to diagnose, prognose, prevent, and/or treatcancers or neoplasms including immune cell or immune tissue-relatedcancers or neoplasms. Examples of cancers or neoplasms that may beprevented, diagnosed, or treated by fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, acute myelogenous leukemia, chronicmyelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acutelymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/ordiseases and disorders described in the section entitled“Hyperproliferative Disorders” elsewhere herein.

[0473] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a therapy for decreasing cellular proliferation ofLarge B-cell Lymphomas.

[0474] In another specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used as a means of decreasing the involvement of B cellsand Ig associated with Chronic Myelogenous Leukemia.

[0475] In specific embodiments, the compositions of the invention areused as an agent to boost immunoresponsiveness among B cellimmunodeficient individuals, such as, for example, an individual who hasundergone a partial or complete splenectomy.

[0476] Blood-Related Disorders

[0477] In a preferred embodiment, albumin fusion proteins of theinvention comprising a Therapeutic protein portion corresponding toimmunoglobulins, serum cholinesterase, alpha-I antitrypsin, aprotinin,and coagulation factors in both pre and active forms (e.g., including,but not limited to, von Willebrand factor, fibrinogen, factor II, factorVII, factor VIIA activated factor, factor VIII, factor IX, factor X,factor XIII, ci inactivator, antithrombin III, thrombin and prothrombin,apo-lipoprotein, c-reactive protein, and protein C) and fragments and/orvariants thereof may be used to modulate hemostatic (the stopping ofbleeding) or thrombolytic (clot dissolving) activity and/or treat,prevent, diagnose, prognose, and/or detect blood-related disorders.

[0478] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused to modulate hemostatic (the stopping of bleeding) or thrombolytic(clot dissolving) activity. For example, by increasing hemostatic orthrombolytic activity, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used to treat or prevent blood coagulation diseases, disorders,and/or conditions (e.g., afibrinogenemia, factor deficiencies,hemophilia), blood platelet diseases, disorders, and/or conditions(e.g., thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention thatcan decrease hemostatic or thrombolytic activity could be used toinhibit or dissolve clotting. These molecules could be important in thetreatment or prevention of heart attacks (infarction), strokes, orscarring.

[0479] In specific embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be used to prevent, diagnose, prognose, and/or treatthrombosis, arterial thrombosis, venous thrombosis, thromboembolism,pulmonary embolism, atherosclerosis, myocardial infarction, transientischemic attack, unstable angina. In specific embodiments, the albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may be used for the prevention ofocculsion of saphenous grafts, for reducing the risk of periproceduralthrombosis as might accompany angioplasty procedures, for reducing therisk of stroke in patients with atrial fibrillation includingnonrheumatic atrial fibrillation, for reducing the risk of embolismassociated with mechanical heart valves and or mitral valves disease.Other uses for the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include, but are not limited to, the prevention of occlusions inextrcorporeal devices (e.g., intravascular canulas, vascular accessshunts in hemodialysis patients, hemodialysis machines, andcardiopulmonary bypass machines).

[0480] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, may be used to prevent, diagnose, prognose, and/or treatdiseases and disorders of the blood and/or blood forming organsassociated with the tissue(s) in which the polypeptide of the inventionis expressed.

[0481] The fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used tomodulate hematopoietic activity (the formation of blood cells). Forexample, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused to increase the quantity of all or subsets of blood cells, such as,for example, erythrocytes, lymphocytes (B or T cells), myeloid cells(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of anemias and leukopenias described below.Alternatively, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused to decrease the quantity of all or subsets of blood cells, such as,for example, erythrocytes, lymphocytes (B or T cells), myeloid cells(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of leukocytoses, such as, for exampleeosinophilia.

[0482] The fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used toprevent, treat, or diagnose blood dyscrasia.

[0483] Anemias are conditions in which the number of red blood cells oramount of hemoglobin (the protein that carries oxygen) in them is belownormal. Anemia may be caused by excessive bleeding, decreased red bloodcell production, or increased red blood cell destruction (hemolysis).The albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating, preventing, and/or diagnosing anemias. Anemias that may betreated prevented or diagnosed by the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include iron deficiency anemia, hypochromic anemia, microcyticanemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquiredsideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g.,pernicious anemia, (vitamin B12 deficiency) and folic acid deficiencyanemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolyticanemia, microangiopathic hemolytic anemia, and paroxysmal nocturnalhemoglobinuria). The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in treating, preventing, and/or diagnosing anemias associatedwith diseases including but not limited to, anemias associated withsystemic lupus erythematosus, cancers, lymphomas, chronic renal disease,and enlarged spleens. The albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful in treating, preventing, and/or diagnosing anemias arisingfrom drug treatments such as anemias associated with methyldopa,dapsone, and/or sulfadrugs. Additionally, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in treating, preventing, and/or diagnosinganemias associated with abnormal red blood cell architecture includingbut not limited to, hereditary spherocytosis, hereditary elliptocytosis,gI ucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.

[0484] The albumin fusion proteins of the invention andiorpolynucleotides encoding albumin fusion proteins of the invention may beuseful in treating, preventing, and/or diagnosing hemoglobinabnormalities, (e.g., those associated with sickle cell anemia,hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease).Additionally, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in diagnosing, prognosing, preventing, and/or treatingthalassemias, including, but not limited to, major and minor forms ofalpha-thalassemia and beta-thalassemia.

[0485] In another embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating bleeding disorders including, but not limited to,thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, andthrombotic thrombocytopenic purpura), Von Willebrand's disease,hereditary platelet disorders (e.g., storage pool disease such asChediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2dysfunction, thromboasthenia, and Bernard-Soulier syndrome),hemolytic-uremic syndrome, hemophelias such as hemophelia A or FactorVII deficiency and Christmas disease or Factor IX deficiency, HereditaryHemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome,allergic purpura (Henoch Schonlein purpura) and disseminatedintravascular coagulation.

[0486] The effect of the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention on theclotting time of blood may be monitored using any of the clotting testsknown in the art including, but not limited to, whole blood partialthromboplastin time (PTT), the activated partial thromboplastin time(aPTT), the activated clotting time (ACT), the recalcified activatedclotting time, or the Lee-White Clotting time.

[0487] Several diseases and a variety of drugs can cause plateletdysfunction. Thus, in a specific embodiment, the albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention may be useful in diagnosing, prognosing, preventing,and/or treating acquired platelet dysfunction such as plateletdysfunction accompanying kidney failure, leukemia, multiple myeloma,cirrhosis of the liver, and systemic lupus erythematosus as well asplatelet dysfunction associated with drug treatments, includingtreatment with aspirin, ticlopidine, nonsteroidal anti-inflammatorydrugs (used for arthritis, pain, and sprains), and penicillin in highdoses.

[0488] In another embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating diseases and disorders characterized by or associated withincreased or decreased numbers of white blood cells. Leukopenia occurswhen the number of white blood cells decreases below normal. Leukopeniasinclude, but are not limited to, neutropenia and lymphocytopenia. Anincrease in the number of white blood cells compared to normal is knownas leukocytosis. The body generates increased numbers of white bloodcells during infection. Thus, leukocytosis may simply be a normalphysiological parameter that reflects infection. Alternatively,leukocytosis may be an indicator of injury or other disease such ascancer. Leokocytoses, include but are not limited to, eosinophilia, andaccumulations of macrophages. In specific embodiments, the albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may be useful in diagnosing,prognosing, preventing, and/or treating leukopenia. In other specificembodiments, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in diagnosing, prognosing, preventing, and/or treatingleukocytosis.

[0489] Leukopenia may be a generalized decreased in all types of whiteblood cells, or may be a specific depletion of particular types of whiteblood cells. Thus, in specific embodiments, the albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention may be useful in diagnosing, prognosing, preventing,and/or treating decreases in neutrophil numbers, known as neutropenia.Neutropenias that may be diagnosed, prognosed, prevented, and/or treatedby the albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention include, but are notlimited to, infantile genetic agranulocytosis, familial neutropenia,cyclic neutropenia, neutropenias resulting from or associated withdietary deficiencies (e.g., vitamin B 12 deficiency or folic aciddeficiency), neutropenias resulting from or associated with drugtreatments (e.g., antibiotic regimens such as penicillin treatment,sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs,anti-thyroid drugs, and cancer chemotherapy), and neutropenias resultingfrom increased neutrophil destruction that may occur in association withsome bacterial or viral infections, allergic disorders, autoimmunediseases, conditions in which an individual has an enlarged spleen(e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatmentregimens.

[0490] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in diagnosing, prognosing, preventing, and/or treatinglymphocytopenias (decreased numbers of B and/or T lymphocytes),including, but not limited to, lymphocytopenias resulting from orassociated with stress, drug treatments (e.g., drug treatment withcorticosteroids, cancer chemotherapies, and/or radiation therapies),AIDS infection and/or other diseases such as, for example, cancer,rheumatoid arthritis, systemic lupus erythematosus, chronic infections,some viral infections and/or hereditary disorders (e.g., DiGeorgesyndrome, Wiskott-Aldrich Syndome, severe combined immunodeficiency,ataxia telangiectsia).

[0491] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful in diagnosing, prognosing, preventing, and/or treating diseasesand disorders associated with macrophage numbers and/or macrophagefunction including, but not limited to, Gaucher's disease, Niemann-Pickdisease, Letterer-Siwe disease and Hand-Schuller-Christian disease.

[0492] In another embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating diseases and disorders associated with eosinophil numbersand/or eosinophil function including, but not limited to, idiopathichypereosinophilic syndrome, eosinophilia-myalgia syndrome, andHand-Schuller-Christian disease.

[0493] In yet another embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating leukemias and lymphomas including, but not limited to, acutelymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic,myelogenous, myeloblastic, or myelomonocytic) leukemia, chroniclymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezarysyndrome, and Hairy cell leukenia), chronic myelocytic (myeloid,myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.

[0494] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in diagnosing, prognosing, preventing, and/ortreating diseases and disorders of plasma cells including, but notlimited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonalgammopathies of undetermined significance, multiple myeloma,macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia,and Raynaud's phenomenon.

[0495] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in treating, preventing, and/or diagnosingmyeloproliferative disorders, including but not limited to, polycythemiavera, relative polycythemia, secondary polycythemia, myelofibrosis,acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia,(including both primary and seconday thrombocythemia) and chronicmyelocytic leukemia.

[0496] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful as a treatment prior to surgery, to increaseblood cell production.

[0497] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful as an agent to enhance the migration,phagocytosis, superoxide production, antibody dependent cellularcytotoxicity of neutrophils, eosionophils and macrophages.

[0498] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful as an agent to increase the number of stem cellsin circulation prior to stem cells pheresis. In another specificembodiment, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beuseful as an agent to increase the number of stem cells in circulationprior to platelet pheresis.

[0499] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful as an agent to increase cytokine production.

[0500] In other embodiments, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be useful in preventing, diagnosing, and/or treatingprimary hematopoietic disorders.

[0501] Hyperproliferative Disorders

[0502] In certain embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused to treat or detect hyperproliferative disorders, includingneoplasms. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayinhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayproliferate other cells which can inhibit the hyperproliferativedisorder.

[0503] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[0504] Examples of hyperproliferative disorders that can be treated ordetected by fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention include, but are notlimited to neoplasms located in the: colon, abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

[0505] Similarly, other hyperproliferative disorders can also be treatedor detected by fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention. Examples of suchhyperproliferative disorders include, but are not limited to: AcuteChildhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, AcuteLymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma,Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer,Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, AdultHodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin'sLymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, IntestinalCancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet CellPancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer,Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

[0506] In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to diagnose, prognose, prevent, and/or treatpremalignant conditions and to prevent progression to a neoplastic ormalignant state, including but not limited to those disorders describedabove. Such uses are indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.)

[0507] Hyperplasia is a form of controlled cell proliferation, involvingan increase in cell number in a tissue or organ, without significantalteration in structure or function. Hyperplastic disorders which can bediagnosed, prognosed, prevented, and/or treated with fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention include, but are not limited to, angiofollicularmediastinal lymph node hyperplasia, angiolymphoid hyperplasia witheosinophilia, a typical melanocytic hyperplasia, basal cell hyperplasia,benign giant lymph node hyperplasia, cementum hyperplasia, congenitaladrenal hyperplasia, congenital sebaceous hyperplasia, cystichyperplasia, cystic hyperplasia of the breast, denture hyperplasia,ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia,focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibroushyperplasia, inflammatory papillary hyperplasia, intravascular papillaryendothelial hyperplasia, nodular hyperplasia of prostate, nodularregenerative hyperplasia, pseudoepitheliomatous hyperplasia, senilesebaceous hyperplasia, and verrucous hyperplasia.

[0508] Metaplasia is a form of controlled cell growth in which one typeof adult or fully differentiated cell substitutes for another type ofadult cell. Metaplastic disorders which can be diagnosed, prognosed,prevented, and/or treated with fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to, agnogenic myeloid metaplasia, apocrinemetaplasia, a typical metaplasia, autoparenchymatous metaplasia,connective tissue metaplasia, epithelial metaplasia, intestinalmetaplasia, metaplastic anemia, metaplastic ossification, metaplasticpolyps, myeloid metaplasia, primary myeloid metaplasia, secondarymyeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion,and symptomatic myeloid metaplasia.

[0509] Dysplasia is frequently a forerunner of cancer, and is foundmainly in the epithelia; it is the most disorderly form ofnon-neoplastic cell growth, involving a loss in individual celluniformity and in the architectural orientation of cells. Dysplasticcells often have abnormally large, deeply stained nuclei, and exhibitpleomorphism. Dysplasia characteristically occurs where there existschronic irritation or inflammation. Dysplastic disorders which can bediagnosed, prognosed, prevented, and/or treated with fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention include, but are not limited to, anhidrotic ectodermaldysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia,atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,cervical dysplasia, chondroectodermal dysplasia, cleidocranialdysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia,craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentindysplasia, diaphysial dyspiasia, ectodermal dysplasia, enamel dysplasia,encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,epithelial dysplasia, faciodigitogenital dysplasia, familial fibrousdysplasia of jaws, familial white folded dysplasia, fibromusculardysplasia, fibrous dysplasia of bone, florid osseous dysplasia,hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammarydysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondinidysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia,multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, ophthalmomandibulomelic dysplasia, pen apical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

[0510] Additional pre-neoplastic disorders which can be diagnosed,prognosed, prevented, and/or treated with fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but are not limited to, benign dysproliferativedisorders (e.g., benign tumors, fibrocystic conditions, tissuehypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia),leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis,and solar keratosis.

[0511] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, may be used to diagnose and/or prognose disorders associatedwith the tissue(s) in which the polypeptide of the invention isexpressed.

[0512] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionconjugated to a toxin or a radioactive isotope, as described herein, maybe used to treat cancers and neoplasms, including, but not limited to,those described herein. In a further preferred embodiment, albuminfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention conjugated to a toxin or a radioactiveisotope, as described herein, may be used to treat acute myelogenousleukemia.

[0513] Additionally, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayaffect apoptosis, and therefore, would be useful in treating a number ofdiseases associated with increased cell survival or the inhibition ofapoptosis. For example, diseases associated with increased cell survivalor the inhibition of apoptosis that could be diagnosed, prognosed,prevented, and/or treated by polynucleotides, polypeptides, and/oragonists or antagonists of the invention, include cancers (such asfollicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection.

[0514] In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to inhibit growth, progression, and/or metastasis of cancers, inparticular those listed above.

[0515] Additional diseases or conditions associated with increased cellsurvival that could be diagnosed, prognosed, prevented, and/or treatedby fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, include, but are not limitedto, progression, and/or metastases of malignancies and related disorderssuch as leukemia (including acute leukemias (e.g., acute lymphocyticleukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0516] Diseases associated with increased apoptosis that could bediagnosed, prognosed, prevented, and/or treated by fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa, cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet'sdisease, Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0517] Hyperproliferative diseases and/or disorders that could bediagnosed, prognosed, prevented, and/or treated by fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, include, but are not limited to, neoplasms located in theliver, abdomen, bone, breast, digestive system, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous system (central andperipheral), lymphatic system, pelvis, skin, soft tissue, spleen,thorax, and urogenital tract.

[0518] Similarly, other hyperproliferative disorders can also bediagnosed, prognosed, prevented, and/or treated by fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention. Examples of such hyperproliferative disorders include,but are not limited to: hypergammaglobulinemia, lymphoproliferativedisorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

[0519] Another preferred embodiment utilizes polynucleotides encodingalbumin fusion proteins of the invention to inhibit aberrant cellulardivision, by gene therapy using the present invention, and/or proteinfusions or fragments thereof.

[0520] Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide encoding an albumin fusion protein of the presentinvention, wherein said polynucleotide represses said expression.

[0521] Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the fusion proteinof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324-326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

[0522] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[0523] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[0524] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[0525] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0526] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[0527] Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention of the presentinvention are useful in inhibiting the angiogenesis of proliferativecells or tissues, either alone, as a protein fusion, or in combinationwith other polypeptides directly or indirectly, as described elsewhereherein. In a most preferred embodiment, said anti-angiogenesis effectmay be achieved indirectly, for example, through the inhibition ofhematopoietic, tumor-specific cells, such as tumor-associatedmacrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53(1998), which is hereby incorporated by reference).

[0528] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful ininhibiting proliferative cells or tissues through the induction ofapoptosis. These fusion protieins and/or polynucleotides may act eitherdirectly, or indirectly to induce apoptosis of proliferative cells andtissues, for example in the activation of a death-domain receptor, suchas tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1),TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-relatedapoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-OsthoffK, et.al., Eur J Biochem 254(3):439-59 (1998), which is herebyincorporated by reference). Moreover, in another preferred embodiment ofthe present invention, these fusion proteins and/or polynucleotides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of these proteins, either alone or in combination with smallmolecule drugs or adjuviants, such as apoptonin, galectins,thioredoxins, anti-inflammatory proteins (See for example, Mutat Res400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem BiolInteract. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998),Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporatedby reference).

[0529] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention are useful ininhibiting the metastasis of proliferative cells or tissues. Inhibitionmay occur as a direct result of administering these albumin fusionproteins and/or polynucleotides, or indirectly, such as activating theexpression of proteins known to inhibit metastasis, for example alpha 4integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, whichis hereby incorporated by reference). Such thereapeutic affects of thepresent invention may be achieved either alone, or in combination withsmall molecule drugs or adjuvants.

[0530] In another embodiment, the invention provides a method ofdelivering compositions containing the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention to targeted cells expressing the a polypeptide bound by, thatbinds to, or associates with an albumin fuison protein of the invention.Albumin fusion proteins of the invention may be associated with withheterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions.

[0531] Albumin fusion proteins of the invention are useful in enhancingthe immunogenicity and/or antigenicity of proliferating cells ortissues, either directly, such as would occur if the albumin fusionproteins of the invention ‘vaccinated’ the immune response to respond toproliferative antigens and immunogens, or indirectly, such as inactivating the expression of proteins known to enhance the immuneresponse (e.g. chemokines), to said antigens and immunogens.

[0532] Renal Disorders

[0533] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be used to treat,prevent, diagnose, and/or prognose disorders of the renal system. Renaldisorders which can be diagnosed, prognosed, prevented, and/or treatedwith compositions of the invention include, but are not limited to,kidney failure, nephritis, blood vessel disorders of kidney, metabolicand congenital kidney disorders, urinary disorders of the kidney,autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, andkidney cancers.

[0534] Kidney diseases which can be diagnosed, prognosed, prevented,and/or treated with compositions of the invention include, but are notlimited to, acute kidney failure, chronic kidney failure, atheroembolicrenal failure, end-stage renal disease, inflammatory diseases of thekidney (e.g., acute glomerulonephritis, postinfectiousglomerulonephritis, rapidly progressive glomerulonephritis, nephroticsyndrome, membranous glomerulonephritis, familial nephrotic syndrome,membranoproliferative glomerulonephritis I and II, mesangialproliferative glomerulonephritis, chronic glomerulonephritis, acutetubulointerstitial nephritis, chronic tubulointerstitial nephritis,acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis,lupus nephritis, chronic nephritis, interstitial nephritis, andpost-streptococcal glomerulonephritis), blood vessel disorders of thekidneys (e.g., kidney infarction, atheroembolic kidney disease, corticalnecrosis, malignant nephrosclerosis, renal vein thrombosis, renalunderperfusion, renal retinopathy, renal ischemia-reperfusion, renalartery embolism, and renal artery stenosis), and kidney disordersresulting form urinary tract disease (e.g., pyelonephritis,hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), refluxnephropathy, urinary tract infections, urinary retention, and acute orchronic unilateral obstructive uropathy.)

[0535] In addition, compositions of the invention can be used todiagnose, prognose, prevent, and/or treat metabolic and congenitaldisorders of the kidney (e.g., uremia, renal amyloidosis, renalosteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenicdiabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocysticosteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle'ssyndrome, polycystic kidney disease, medullary cystic disease, medullarysponge kidney, Alport's syndrome, nail-patella syndrome, congenitalnephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabeticnephropathy, nephrogenic diabetes insipidus, analgesic nephropathy,kidney stones, and membranous nephropathy), and autoimmune disorders ofthe kidney (e.g., systemic lupus erythematosus (SLE), Goodpasturesyndrome, IgA nephropathy, and IgM mesangial proliferativeglomerulonephritis).

[0536] Compositions of the invention can also be used to diagnose,prognose, prevent, and/or treat sclerotic or necrotic disorders of thekidney (e.g., glomeruloscierosis, diabetic nephropathy, focal segmentalglomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renalpapillary necrosis), cancers of the kidney (e.g., nephroma,hypernephroma, nephroblastoma, renal cell cancer, transitional cellcancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor),and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema,hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia,hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, andhyperphosphatemia).

[0537] Compositions of the invention may be administered using anymethod known in the art, including, but not limited to, direct needleinjection at the delivery site, intravenous injection, topicaladministration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. Compositions of theinvention may be administered as part of a Therapeutic, described inmore detail below. Methods of delivering polynucleotides of theinvention are described in more detail herein.

[0538] Cardiovascular Disorders

[0539] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be used to treat,prevent, diagnose, and/or prognose cardiovascular disorders, including,but not limited to, peripheral artery disease, such as limb ischemia.

[0540] Cardiovascular disorders include, but are not limited to,cardiovascular abnormalities, such as arterio-arterial fistula,arterioyenous fistula, cerebral arterioyenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include, but are not limited to, aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

[0541] Cardiovascular disorders also include, but are not limited to,heart disease, such as arrhythmias, carcinoid heart disease, highcardiac output, low cardiac output, cardiac tamponade, endocarditis(including bacterial), heart aneurysm, cardiac arrest, congestive heartfailure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema,heart hypertrophy, congestive cardiomyopathy, left ventricularhypertrophy, right ventricular hypertrophy, post-infarction heartrupture, ventricular septal rupture, heart valve diseases, myocardialdiseases, myocardial ischemia, pericardial effusion, pericarditis(including constrictive and tuberculous), pneumopericardium,postpericardiotomy syndrome, pulmonary heart disease, rheumatic heartdisease, ventricular dysfunction, hyperemia, cardiovascular pregnancycomplications, Scimitar Syndrome, cardiovascular syphilis, andcardiovascular tuberculosis.

[0542] Arrhythmias include, but are not limited to, sinus arrhythmia,atrial fibrillation, atrial flutter, bradycardia, extrasystole,Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QTsyndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-typepre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinussyndrome, tachycardias, and ventricular fibrillation. Tachycardiasinclude paroxysmal tachycardia, supraventricular tachycardia,accelerated idioventricular rhythm, atrioventricular nodal reentrytachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia,sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades dePointes, and ventricular tachycardia.

[0543] Heart valve diseases include, but are not limited to, aorticvalve insufficiency, aortic valve stenosis, hear murmurs, aortic valveprolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valveinsufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valveinsufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspidvalve insufficiency, and tricuspid valve stenosis.

[0544] Myocardial diseases include, but are not limited to, alcoholiccardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy,aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictivecardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury,and myocarditis.

[0545] Myocardial ischemias include, but are not limited to, coronarydisease, such as angina pectoris, coronary aneurysm, coronaryarteriosclerosis, coronary thrombosis, coronary vasospasm, myocardialinfarction and myocardial stunning.

[0546] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0547] Aneurysms include, but are not limited to, dissecting aneurysms,false aneurysms, infected aneurysms, ruptured aneurysms, aorticaneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, andiliac aneurysms.

[0548] Arterial occlusive diseases include, but are not limited to,arteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease, renal artery obstruction, retinal artery occlusion, andthromboangiitis obliterans.

[0549] Cerebrovascular disorders include, but are not limited to,carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arterioyenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

[0550] Embolisms include, but are not limited to, air embolisms,amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fatembolisms, pulmonary embolisms, and thromoboembolisms. Thrombosisinclude, but are not limited to, coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

[0551] Ischemic disorders include, but are not limited to, cerebralischemia, ischemic colitis, compartment syndromes, anterior compartmentsyndrome, myocardial ischemia, reperfusion injuries, and peripheral limbischemia. Vasculitis includes, but is not limited to, aortitis,arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneouslymph node syndrome, thromboangiitis obliterans, hypersensitivityvasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis,and Wegener's granulomatosis.

[0552] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be administeredusing any method known in the art, including, but not limited to, directneedle injection at the delivery site, intravenous injection, topicaladministration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. Methods of deliveringpolynucleotides are described in more detail herein.

[0553] Respiratory Disorders

[0554] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be used to treat,prevent, diagnose, and/or prognose diseases and/or disorders of therespiratory system.

[0555] Diseases and disorders of the respiratory system include, but arenot limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acuterhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis),nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the noseand juvenile papillomas, vocal cord polyps, nodules (singer's nodules),contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g.,viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngealabscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer ofthe nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,squamous cell carcinoma, small cell (oat cell) carcinoma, large cellcarcinoma, and adenocarcinoma), allergic disorders (eosinophilicpneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergicalveolitis, allergic interstitial pneumonitis, organic dustpneumoconiosis, allergic bronchopulmonary aspergillosis, asthma,Wegener's granulomatosis (granulomatous vasculitis), Goodpasture'ssyndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcuspneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus(staphylococcal pneumonia), Gram-negative bacterial pneumonia (causedby, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniaepneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila(Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), andviral pneumonia (e.g., influenza, chickenpox (varicella).

[0556] Additional diseases and disorders of the respiratory systeminclude, but are not limited to bronchiolitis, polio (poliomyelitis),croup, respiratory syncytial viral infection, mumps, erythemainfectiosum (fifth disease), roseola infantum, progressive rubellapanencephalitis, german measles, and subacute sclerosingpanencephalitis), fungal pneumonia (e.g., Histoplasmosis,Coccidioidomycosis, Blastomycosis, fungal infections in people withseverely suppressed immune systems (e.g., cryptococcosis, caused byCryptococcus neoformans; aspergillosis, caused by Aspergillus spp.;candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii(pneumocystis pneumonia), a typical pneumonias (e.g., Mycoplasma andChiamydia spp.), opportunistic infection pneumonia, nosocomialpneumonia, chemical pneumonitis, and aspiration pneumonia, pleuraldisorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g.,simple spontaneous pneumothorax, complicated spontaneous pneumothorax,tension pneumothorax)), obstructive airway diseases (e.g., asthma,chronic obstructive pulmonary disease (COPD), emphysema, chronic oracute bronchitis), occupational lung diseases (e.g., silicosis, blacklung (coal workers' pneumoconiosis), asbestosis, berylliosis,occupational asthsma, byssinosis, and benign pneumoconioses),Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosingalveolitis, usual interstitial pneumonia), idiopathic pulmonaryfibrosis, desquamative interstitial pneumonia, lymphoid interstitialpneumonia, histiocytosis X (e.g., Letterer-Siwe disease,Hand-Schtiller-Christian disease, eosinophilic granuloma), idiopathicpulmonary hemosiderosis, sarcoidosis and pulmonary alveolarproteinosis), Acute respiratory distress syndrome (also called, e.g.,adult respiratory distress syndrome), edema, pulmonary embolism,bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lungabscess (caused by, e.g., Staphylococcus aureus or Legionellapneumophila), and cystic fibrosis.

[0557] Anti-Angiogenesis Activity

[0558] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

[0559] The present invention provides for treatment of diseases ordisorders associated with neovascularization by administration of fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention. Malignant and metastatic conditions which canbe treated with the polynucleotides and polypeptides, or agonists orantagonists of the invention include, but are not limited to,malignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, thepresent invention provides a method of treating an angiogenesis-relateddisease and/or disorder, comprising administering to an individual inneed thereof a therapeutically effective amount of an albumin fusionprotein of the invention and/or polynucleotides encoding an albuminfusion protein of the invention. For example, fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, but are not limited tosolid tumors, including prostate, lung, breast, ovarian, stomach,pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroidcancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi'ssarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer;advanced malignancies; and blood born tumors such as leukemias. Forexample, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be deliveredtopically, in order to treat cancers such as skin cancer, head and necktumors, breast tumors, and Kaposi's sarcoma.

[0560] Within yet other aspects, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beutilized to treat superficial forms of bladder cancer by, for example,intravesical administration. Albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be delivered directly into the tumor, or near the tumor site, viainjection or a catheter. Of course, as the artisan of ordinary skillwill appreciate, the appropriate mode of administration will varyaccording to the cancer to be treated. Other modes of delivery arediscussed herein.

[0561] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be useful intreating other disorders, besides cancers, which involve angiogenesis.These disorders include, but are not limited to: benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases,for example, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormalblood vessel growth) of the eye; rheumatoid arthritis; psoriasis;delayed wound healing; endometriosis; vasculogenesis; granulations;hypertrophic scars (keloids); nonunion fractures; scieroderma; trachoma;vascular adhesions; myocardial angiogenesis; coronary collaterals;cerebral collaterals; arterioyenous malformations; ischemic limbangiogenesis; Osler-Webber Syndrome; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; fibromusculardysplasia; wound granulation; Crohn's disease; and atherosclerosis.

[0562] For example, within one aspect of the present invention methodsare provided for treating hypertrophic scars and keloids, comprising thestep of administering albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention to ahypertrophic scar or keloid.

[0563] Within one embodiment of the present invention fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention are directly injected into a hypertrophic scar or keloid,in order to prevent the progression of these lesions. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development. As notedabove, the present invention also provides methods for treatingneovascular diseases of the eye, including for example, cornealneovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

[0564] Moreover, Ocular disorders associated with neovascularizationwhich can be treated with the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventioninclude, but are not limited to: neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

[0565] Thus, within one aspect of the present invention methods areprovided for treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (e.g., fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention) to the cornea, such that the formation of blood vessels isinhibited. Briefly, the cornea is a tissue which normally lacks bloodvessels. In certain pathological conditions however, capillaries mayextend into the cornea from the pericorneal vascular plexus of thelimbus. When the cornea becomes vascularized, it also becomes clouded,resulting in a decline in the patient's visual acuity. Visual loss maybecome complete if the cornea completely opacitates. A wide variety ofdisorders can result in corneal neovascularization, including forexample, corneal infections (e.g., trachoma, herpes simplex keratitis,leishmaniasis and onchocerciasis), immunological processes (e.g., graftrejection and Stevens-Johnson's syndrome), alkali burns, trauma,inflammation (of any cause), toxic and nutritional deficiency states,and as a complication of wearing contact lenses.

[0566] Within particularly preferred embodiments of the invention, maybe prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

[0567] Within other embodiments, the compounds described above may beinjected directly into the corneal stroma by an ophthalmologist undermicroscopic guidance. The preferred site of injection may vary with themorphology of the individual lesion, but the goal of the administrationwould be to place the composition at the advancing front of thevasculature (i.e., interspersed between the blood vessels and the normalcornea). In most cases this would involve perilimbic corneal injectionto “protect” the cornea from the advancing blood vessels. This methodmay also be utilized shortly after a corneal insult in order toprophylactically prevent corneal neovascularization. In this situationthe material could be injected in the perilimbic cornea interspersedbetween the corneal lesion and its undesired potential limbic bloodsupply. Such methods may also be utilized in a similar fashion toprevent capillary invasion of transplanted corneas. In asustained-release form injections might only be required 2-3 times peryear. A steroid could also be added to the injection solution to reduceinflammation resulting from the injection itself.

[0568] Within another aspect of the present invention, methods areprovided for treating neovascular glaucoma, comprising the step ofadministering to a patient a therapeutically effective amount of analbumin fusion protein of the invention and/or polynucleotides encodingan albumin fusion protein of the invention to the eye, such that theformation of blood vessels is inhibited. In one embodiment, the compoundmay be administered topically to the eye in order to treat early formsof neovascular glaucoma. Within other embodiments, the compound may beimplanted by injection into the region of the anterior chamber angle.Within other embodiments, the compound may also be placed in anylocation such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of an albumin fusion protein of the invention and/orpolynucleotides encoding an albumin fusion protein of the invention tothe eyes, such that the formation of blood vessels is inhibited.

[0569] Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

[0570] Within another aspect of the present invention, methods areprovided for treating retrolental fibroplasia, comprising the step ofadministering to a patient a therapeutically effective amount of analbumin fusion protein of the invention and/or polynucleotides encodingan albumin fusion protein of the invention to the eye, such that theformation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

[0571] Additionally, disorders which can be treated with fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention include, but are not limited to, hemangioma, arthritis,psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions.

[0572] Moreover, disorders and/or states, which can be treated,prevented, diagnosed, and/or prognosed with the the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention of the invention include, but are not limitedto, solid tumors, blood born tumors such as leukemias, tumor metastasis,Kaposi's sarcoma, benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoidarthritis, psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arterioyenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0573] In one aspect of the birth control method, an amount of thecompound sufficient to block embryo implantation is administered beforeor after intercourse and fertilization have occurred, thus providing aneffective method of birth control, possibly a “morning after” method.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention may also be used in controllingmenstruation or administered as either a peritoneal lavage fluid or forperitoneal implantation in the treatment of endometriosis.

[0574] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be incorporatedinto surgical sutures in order to prevent stitch granulomas.

[0575] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may be utilized in awide variety of surgical procedures. For example, within one aspect ofthe present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

[0576] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administeringalbumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention to the resection margins of atumor subsequent to excision, such that the local recurrence of cancerand the formation of new blood vessels at the site is inhibited. Withinone embodiment of the invention, the anti-angiogenic compound isadministered directly to the tumor excision site (e.g., applied byswabbing, brushing or otherwise coating the resection margins of thetumor with the anti-angiogenic compound). Alternatively, theanti-angiogenic compounds may be incorporated into known surgical pastesprior to administration. Within particularly preferred embodiments ofthe invention, the anti-angiogenic compounds are applied after hepaticresections for malignancy, and after neurosurgical operations.

[0577] Within one aspect of the present invention, fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention may be administered to the resection margin of a widevariety of tumors, including for example, breast, colon, brain andhepatic tumors. For example, within one embodiment of the invention,anti-angiogenic compounds may be administered to the site of aneurological tumor subsequent to excision, such that the formation ofnew blood vessels at the site are inhibited.

[0578] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention mayalso be administered along with other anti-angiogenic factors.Representative examples of other anti-angiogenic factors include:Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel,Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1, PlasminogenActivator Inhibitor-2, and various forms of the lighter “d group”transition metals.

[0579] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0580] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[0581] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[0582] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, (1992)); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

[0583] Diseases at the Cellular Level

[0584] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated, prevented, diagnosed,and/or prognosed using fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention,include cancers (such as follicular lymphomas, carcinomas with p53mutations, and hormone-dependent tumors, including, but not limited tocolon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection.

[0585] In preferred embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above.

[0586] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endothel iosarcoma, I ymphangiosarcoma,lymphangioendotheliosarcoma, synovi oma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0587] Diseases associated with increased apoptosis that could betreated, prevented, diagnosed, and/or prognesed using fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, include, but are not limited to, AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration andbrain tumor or prior associated disease); autoimmune disorders (such as,multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

[0588] Wound Healing and Epithelial Cell Proliferation

[0589] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, for therapeutic purposes, for example, to stimulateepithelial cell proliferation and basal keratinocytes for the purpose ofwound healing, and to stimulate hair follicle production and healing ofdermal wounds. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe clinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, burns resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associated withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. Albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, could be used to promote dermal reestablishment subsequent todermal loss

[0590] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used toincrease the adherence of skin grafts to a wound bed and to stimulatere-epithelialization from the wound bed. The following are types ofgrafts that fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, can be used to promote skin strength and toimprove the appearance of aged skin.

[0591] It is believed that fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, willalso produce changes in hepatocyte proliferation, and epithelial cellproliferation in the lung, breast, pancreas, stomach, small intestine,and large intestine. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, and gastrointestinal tract. Albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

[0592] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could also be used toreduce the side effects of gut toxicity that result from radiation,chemotherapy treatments or viral infections. Albumin fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, may have a cytoprotective effect on the small intestinemucosa. Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may also stimulatehealing of mucositis (mouth ulcers) that result from chemotherapy andviral infections.

[0593] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could further be usedin full regeneration of skin in full and partial thickness skin defects,including burns, (i.e., repopulation of hair follicles, sweat glands,and sebaceous glands), treatment of other skin defects such aspsoriasis. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could also be used to treatgastric and doudenal ulcers and help heal by scar formation of themucosal lining and regeneration of glandular mucosa and duodenal mucosallining more rapidly. Inflammatory bowel diseases, such as Crohn'sdisease and ulcerative colitis, are diseases which result in destructionof the mucosal surface of the small or large intestine, respectively.Thus, fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent progression of inflammatory bowel disease. Treatment with fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention, is expected to have a significant effect onthe production of mucus throughout the gastrointestinal tract and couldbe used to protect the intestinal mucosa from injurious substances thatare ingested or following surgery. Albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, could be used to treat diseases associate with the underexpression.

[0594] Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could be used toprevent and heal damage to the lungs due to various pathological states.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, which could stimulateproliferation and differentiation and promote the repair of alveoli andbrochiolar epithelium to prevent or treat acute or chronic lung damage.For example, emphysema, which results in the progressive loss of aveoli,and inhalation injuries, i.e., resulting from smoke inhalation andburns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated using polynucleotides or polypeptides,agonists or antagonists of the present invention. Also fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

[0595] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, could stimulate theproliferation and differentiation of hepatocytes and, thus could be usedto alleviate or treat liver diseases and pathologies such as fulminantliver failure caused by cirrhosis, liver damage caused by viralhepatitis and toxic substances (i.e., acetaminophen, carbontetraholoride and other hepatotoxins known in the art).

[0596] In addition, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldbe used treat or prevent the onset of diabetes mellitus. In patientswith newly diagnosed Types I and II diabetes, where some islet cellfunction remains, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, couldbe used to maintain the islet function so as to alleviate, delay orprevent permanent manifestation of the disease. Also, fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention, could be used as an auxiliary in islet celltransplantation to improve or promote islet cell function.

[0597] Neural Activity and Neurological Diseases

[0598] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused for the diagnosis and/or treatment of diseases, disorders, damageor injury of the brain and/or nervous system. Nervous system disordersthat can be treated with the compositions of the invention (e.g., fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention), include, but are not limited to, nervoussystem injuries, and diseases or disorders which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated in a patient(including human and non-human mammalian patients) according to themethods of the invention, include but are not limited to, the followinglesions of either the central (including spinal cord, brain) orperipheral nervous systems: (1) ischemic lesions, in which a lack ofoxygen in a portion of the nervous system results in neuronal injury ordeath, including cerebral infarction or ischemia, or spinal cordinfarction or ischemia; (2) traumatic lesions, including lesions causedby physical injury or associated with surgery, for example, lesionswhich sever a portion of the nervous system, or compression injuries;(3) malignant lesions, in which a portion of the nervous system isdestroyed or injured by malignant tissue which is either a nervoussystem associated malignancy or a malignancy derived from non-nervoussystem tissue; (4) infectious lesions, in which a portion of the nervoussystem is destroyed or injured as a result of infection, for example, byan abscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, or syphilis; (5) degenerative lesions, in which a portionof the nervous system is destroyed or injured as a result of adegenerative process including but not limited to, degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases or disorders, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including, but not limited to, vitamin B12deficiency, folic acid deficiency, Wemicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

[0599] In one embodiment, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to protect neural cells from the damaging effects of hypoxia.In a further preferred embodiment, the albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the compositions of theinvention are used to treat or prevent neural cell injury associatedwith cerebral hypoxia. In one non-exclusive aspect of this embodiment,the albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, are used to treat orprevent neural cell injury associated with cerebral ischemia. In anothernon-exclusive aspect of this embodiment, the albumin fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention are used to treat or prevent neural cell injury associatedwith cerebral infarction.

[0600] In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent neural cell injury associatedwith a stroke. In a specific embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent cerebral neural cell injuryassociated with a stroke.

[0601] In another preferred embodiment, albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention are used to treat or prevent neural cell injury associatedwith a heart attack. In a specific embodiment, albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention are used to treat or prevent cerebral neural cellinjury associated with a heart attack.

[0602] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture either in the presence or absence of hypoxia or hypoxicconditions; (2) increased sprouting of neurons in culture or in vivo;(3) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, in Zhang et al., ProcNatl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci.,10:3507-15 (1990); increased sprouting of neurons may be detected bymethods known in the art, such as, for example the methods set forth inPestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann.Rev. Neurosci., 4:17-42 (1981); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., using techniquesknown in the art and depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

[0603] In specific embodiments, motor neuron disorders that may betreated according to the invention include, but are not limited to,disorders such as infarction, infection, exposure to toxin, trauma,surgical damage, degenerative disease or malignancy that may affectmotor neurons as well as other components of the nervous system, as wellas disorders that selectively affect neurons such as amyotrophic lateralsclerosis, and including, but not limited to, progressive spinalmuscular atrophy, progressive bulbar palsy, primary lateral sclerosis,infantile and juvenile muscular atrophy, progressive bulbar paralysis ofchildhood (Fazio-Londe syndrome), poliomyelitis and the post poliosyndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-ToothDisease).

[0604] Further, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may play a role inneuronal survival; synapse formation; conductance; neuraldifferentiation, etc. Thus, compositions of the invention (includingfusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention) may be used to diagnose and/or treator prevent diseases or disorders associated with these roles, including,but not limited to, learning and/or cognition disorders. Thecompositions of the invention may also be useful in the treatment orprevention of neurodegenerative disease states and/or behaviouraldisorders. Such neurodegenerative disease states and/or behavioraldisorders include, but are not limited to, Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,panic disorder, learning disabilities, ALS, psychoses, autism, andaltered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, compositions of the invention mayalso play a role in the treatment, prevention and/or detection ofdevelopmental disorders associated with the developing embryo, orsexually-linked disorders.

[0605] Additionally, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, maybe useful in protecting neural cells from diseases, damage, disorders,or injury, associated with cerebrovascular disorders including, but notlimited to, carotid artery diseases (e.g., carotid artery thrombosis,carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy,cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarterioyenous malformations, cerebral artery diseases, cerebral embolismand thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, orWallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subduralhematoma, or subarachnoid hemorrhage), cerebral infarction, cerebralischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome,or vertebrobasilar insufficiency), vascular dementia (e.g.,multi-infarct), leukomalacia, periventricular, and vascular headache(e.g., cluster headache or migraines).

[0606] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention, for therapeutic purposes, for example, to stimulateneurological cell proliferation and/or differentiation. Therefore,fusion proteins of the invention and/or polynucleotides encoding albuminfusion proteins of the invention may be used to treat and/or detectneurologic diseases. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used as a marker or detector of a particular nervous system diseaseor disorder.

[0607] Examples of neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include, brain diseases, suchas metabolic brain diseases which includes phenylketonuria such asmaternal phenylketonuria, pyruvate carboxylase deficiency, pyruvatedehydrogenase complex deficiency, Wernicke's Encephalopathy, brainedema, brain neoplasms such as cerebellar neoplasms which includeinfratentorial neoplasms, cerebral ventricle neoplasms such as choroidplexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms,canavan disease, cerebellar diseases such as cerebellar ataxia whichinclude spinocerebellar degeneration such as ataxia telangiectasia,cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis.

[0608] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include cerebrovasculardisorders (such as carotid artery diseases which include carotid arterythrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloidangiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arterioyenous malformations, cerebral arterydiseases, cerebral embolism and thrombosis such as carotid arterythrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebralhemorrhage such as epidural hematoma, subdural hematoma and subarachnoidhemorrhage, cerebral infarction, cerebral ischemia such as transientcerebral ischemia, Subclavian Steal Syndrome and vertebrobasilarinsufficiency, vascular dementia such as multi-infarct dementia,periventricular leukomalacia, vascular headache such as cluster headacheand migraine.

[0609] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include dementia such as AIDSDementia Complex, presenile dementia such as Alzheimer's Disease andCreutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Diseaseand progressive supranuclear palsy, vascular dementia such asmulti-infarct dementia, encephalitis which include encephalitisperiaxialis, viral encephalitis such as epidemic encephalitis, JapaneseEncephalitis, St. Louis Encephalitis, tick-borne encephalitis and WestNile Fever, acute disseminated encephalomyelitis, meningoencephalitissuch as uveomeningoencephalitic syndrome, Postencephalitic ParkinsonDisease and subacute sclerosing panencephalitis, encephalomalacia suchas periventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

[0610] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include hydrocephalus such asDandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamicdiseases such as hypothalamic neoplasms, cerebral malaria, narcolepsywhich includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor,Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebraltoxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, centralnervous system infections such as AIDS Dementia Complex, Brain Abscess,subdural empyema, encephalomyelitis such as Equine Encephalomyelitis,Venezuelan Equine Encephalomyelitis, Necrotizing HemorrhagicEncephalomyelitis, Visna, and cerebral malaria.

[0611] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include meningitis such asarachnoiditis, aseptic meningtitis such as viral meningtitis whichincludes lymphocytic choriomeningitis, Bacterial meningtitis whichincludes Haemophilus Meningtitis, Listeria Meningtitis, MeningococcalMeningtitis such as Waterhouse-Friderichsen Syndrome, PneumococcalMeningtitis and meningeal tuberculosis, fungal meningitis such asCryptococcal Meningtitis, subdural effusion, meningoencephalitis such asuvemeningoencephalitic syndrome, myelitis such as transverse myelitis,neurosyphilis such as tabes dorsalis, poliomyelitis which includesbulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases(such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy,Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebraltoxoplasmosis.

[0612] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include central nervous systemneoplasms such as brain neoplasms that include cerebellar neoplasms suchas infratentorial neoplasms, cerebral ventricle neoplasms such aschoroid plexus neoplasms, hypothalamic neoplasms and supratentorialneoplasms, meningeal neoplasms, spinal cord neoplasms which includeepidural neoplasms, demyelinating diseases such as Canavan Diseases,diffuse cerebral sceloris which includes adrenoleukodystrophy,encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebralsclerosis such as metachromatic leukodystrophy, allergicencephalomyelitis, necrotizing hemorrhagic encephalomyelitis,progressive multifocal leukoencephalopathy, multiple sclerosis, centralpontine myelinolysis, transverse myelitis, neuromyelitis optica,Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High PressureNervous Syndrome, Meningism, spinal cord diseases such as amyotoniacongenita, amyotrophic lateral sclerosis, spinal muscular atrophy suchas Werdnig-Hoffmann Disease, spinal cord compression, spinal cordneoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis,Stiff-Man Syndrome, mental retardation such as Angelman Syndrome,Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidosessuch as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease,Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome,Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such asfucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome,phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome,Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGRSyndrome, nervous system abnormalities such as holoprosencephaly, neuraltube defects such as anencephaly which includes hydrangencephaly,Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele,spinal dysraphism such as spina bifida cystica and spina bifida occulta.

[0613] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include hereditary motor andsensory neuropathies which include Charcot-Marie Disease, Hereditaryoptic atrophy, Refsum's Disease, hereditary spastic paraplegia,Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathiessuch as Congenital Analgesia and Familial Dysautonomia, Neurologicmanifestations (such as agnosia that include Gerstmann's Syndrome,Amnesia such as retrograde amnesia, apraxia, neurogenic bladder,cataplexy, communicative disorders such as hearing disorders thatincludes deafness, partial hearing loss, loudness recruitment andtinnitus, language disorders such as aphasia which include agraphia,anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as AcquiredDyslexia, language development disorders, speech disorders such asaphasia which includes anomia, broca aphasia and Wernicke Aphasia,articulation disorders, communicative disorders such as speech disorderswhich include dysarthria, echolalia, mutism and stuttering, voicedisorders such as aphonia and hoarseness, decerebrate state, delirium,fasciculation, hallucinations, meningism, movement disorders such asangelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia,muscle hypotonia, myoclonus, tic, torticollis and tremor, musclehypertonia such as muscle rigidity such as stiff-man syndrome, musclespasticity, paralysis such as facial paralysis which includes HerpesZoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such asdiplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressiveexternal ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis,Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,quadriplegia, respiratory paralysis and vocal cord paralysis, paresis,phantom limb, taste disorders such as ageusia and dysgeusia, visiondisorders such as amblyopia, blindness, color vision defects, diplopia,hemianopsia, scotoma and subnormal vision, sleep disorders such ashypersomnia which includes Kleine-Levin Syndrome, insomnia, andsomnambulism, spasm such as trismus, unconsciousness such as coma,persistent vegetative state and syncope and vertigo, neuromusculardiseases such as amyotonia congenita, amyotrophic lateral sclerosis,Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscularatrophy such as spinal muscular atrophy, Charcot-Marie Disease andWerdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, MuscularDystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita,Nemaline Myopathy, Familial Periodic Paralysis, MultiplexParamyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome,peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as FacialNeuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders whichincludes amblyopia, nystagmus, oculomotor nerve paralysis,ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome, ChronicProgressive External Ophthalmoplegia which includes Kearns Syndrome,Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis,Optic Nerve Diseases such as Optic Atrophy which includes HereditaryOptic Atrophy, Optic Disk Drusen, Optic Neuritis such as NeuromyelitisOptica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis,Demyelinating Diseases such as Neuromyelitis Optica and Swayback, andDiabetic neuropathies such as diabetic foot.

[0614] Additional neurologic diseases which can be treated or detectedwith fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention include nerve compressionsyndromes such as carpal tunnel syndrome, tarsal tunnel syndrome,thoracic outlet syndrome such as cervical rib syndrome, ulnar nervecompression syndrome, neuralgia such as causalgia, cervico-brachialneuralgia, facial neuralgia and trigeminal neuralgia, neuritis such asexperimental alleroic neuriitis, polyneuritis, polyradiculoneuritis andradiculities such as polyradiculitis, hereditary motor and sensoryneuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy,Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-HoffmannDisease, Hereditary Sensory and Autonomic Neuropathies which includeCongenital Analgesia and Familial Dysautonomia, POEMS Syndrome,Sciatica, Gustatory Sweating and Tetany).

[0615] Endocrine Disorders

[0616] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be used to treat,prevent, diagnose, and/or prognose disorders and/or diseases related tohormone imbalance, and/or disorders or diseases of the endocrine system.

[0617] Hormones secreted by the glands of the endocrine system controlphysical growth, sexual function, metabolism, and other functions.Disorders may be classified in two ways: disturbances in the productionof hormones, and the inability of tissues to respond to hormones. Theetiology of these hormone imbalance or endocrine system diseases,disorders or conditions may be genetic, somatic, such as cancer and someautoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins),or infectious. Moreover, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as a marker or detector of a particular disease or disorder relatedto the endocrine system and/or hormone imbalance.

[0618] Endocrine system and/or hormone imbalance and/or diseasesencompass disorders of uterine motility including, but not limited to:complications with pregnancy and labor (e.g., pre-term labor, post-termpregnancy, spontaneous abortion, and slow or stopped labor); anddisorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea andendometriosis).

[0619] Endocrine system and/or hormone imbalance disorders and/ordiseases include disorders and/or diseases of the pancreas, such as, forexample, diabetes mellitus, diabetes insipidus, congenital pancreaticagenesis, pheochromocytoma—islet cell tumor syndrome; disorders and/ordiseases of the adrenal glands such as, for example, Addison's Disease,corticosteroid deficiency, virilizing disease, hirsutism, Cushing'sSyndrome, hyperaldosteronism, pheochromocytoma; disorders and/ordiseases of the pituitary gland, such as, for example, hyperpituitarism,hypopituitarism, pituitary dwarfism, pituitary adenoma,panhypopituitarism, acromegaly, gigantism; disorders and/or diseases ofthe thyroid, including but not limited to, hyperthyroidism,hypothyroidism, Plummer's disease, Graves' disease (toxic diffusegoiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis,subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis),Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormonecoupling defect, thymic aplasia, Hurthle cell tumours of the thyroid,thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma;disorders and/or diseases of the parathyroid, such as, for example,hyperparathyroidism, hypoparathyroidism; disorders and/or diseases ofthe hypothalamus.

[0620] In addition, endocrine system and/or hormone imbalance disordersand/or diseases may also include disorders and/or diseases of the testesor ovaries, including cancer. Other disorders and/or diseases of thetestes or ovaries further include, for example, ovarian cancer,polycystic ovary syndrome, Klinefelter's syndrome, vanishing testessyndrome (bilateral anorchia), congenital absence of Leydig's cells,cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillaryhaemangioma of the testis (benign), neoplasias of the testis andneo-testis.

[0621] Moreover, endocrine system and/or hormone imbalance disordersand/or diseases may also include disorders and/or diseases such as, forexample, polyglandular deficiency syndromes, pheochromocytoma,neuroblastoma, multiple Endocrine neoplasia, and disorders and/orcancers of endocrine tissues.

[0622] In another embodiment, albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of theinvention, may be used to diagnose, prognose, prevent, and/or treatendocrine diseases and/or disorders associated with the tissue(s) inwhich the Therapeutic protein corresponding to the Therapeutic proteinportion of the albumin protein of the invention is expressed,

[0623] Reproductive System Disorders

[0624] The albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention may beused for the diagnosis, treatment, or prevention of diseases and/ordisorders of the reproductive system. Reproductive system disorders thatcan be treated by the compositions of the invention, include, but arenot limited to, reproductive system injuries, infections, neoplasticdisorders, congenital defects, and diseases or disorders which result ininfertility, complications with pregnancy, labor, or parturition, andpostpartum difficulties.

[0625] Reproductive system disorders and/or diseases include diseasesand/or disorders of the testes, including testicular atrophy, testicularfeminization, cryptorchism (unilateral and bilateral), anorchia, ectopictestis, epididymitis and orchitis (typically resulting from infectionssuch as, for example, gonorrhea, mumps, tuberculosis, and syphilis),testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas,embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sactumors, and teratomas), stromal tumors (e.g., Leydig cell tumors),hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, anddisorders of sperm production (e.g., immotile cilia syndrome, aspermia,asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).

[0626] Reproductive system disorders also include disorders of theprostate gland, such as acute non-bacterial prostatitis, chronicnon-bacterial prostatitis, acute bacterial prostatitis, chronicbacterial prostatitis, prostatodystonia, prostatosis, granulomatousprostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia,and prostate neoplastic disorders, including adenocarcinomas,transitional cell carcinomas, ductal carcinomas, and squamous cellcarcinomas.

[0627] Additionally, the compositions of the invention may be useful inthe diagnosis, treatment, and/or prevention of disorders or diseases ofthe penis and urethra, including inflammatory disorders, such asbalanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis,syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis,chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome,condyloma acuminatum, condyloma latum, and pearly penile papules;urethral abnormalities, such as hypospadias, epispadias, and phimosis;premalignant lesions, including Erythroplasia of Queyrat, Bowen'sdisease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, andvarrucous carcinoma; penile cancers, including squamous cell carcinomas,carcinoma in situ, verrucous carcinoma, and disseminated penilecarcinoma; urethral neoplastic disorders, including penile urethralcarcinoma, bulbomembranous urethral carcinoma, and prostatic urethralcarcinoma; and erectile disorders, such as priapism, Peyronie's disease,erectile dysfunction, and impotence.

[0628] Moreover, diseases and/or disorders of the vas deferens includevasculititis and CBAVD (congenital bilateral absence of the vasdeferens); additionally, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used in the diagnosis, treatment, and/or prevention of diseasesand/or disorders of the seminal vesicles, including hydatid disease,congenital chloride diarrhea, and polycystic kidney disease.

[0629] Other disorders and/or diseases of the male reproductive systeminclude, for example, Klinefelter's syndrome, Young's syndrome,premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener'ssyndrome, high fever, multiple sclerosis, and gynecomastia.

[0630] Further, the polynucleotides, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used in the diagnosis, treatment, and/or prevention of diseasesand/or disorders of the vagina and vulva, including bacterial vaginosis,candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale,lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma,vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonasvaginitis, condyloma acuminatum, syphilis, molluscum contagiosum,atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus,vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvarvestibulitis, and neoplastic disorders, such as squamous cellhyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas,cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.

[0631] Disorders and/or diseases of the uterus include dysmenorrhea,retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatorybleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman'ssyndrome, premature menopause, precocious puberty, uterine polyps,dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals),and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, andsarcomas. Additionally, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be useful as a marker or detector of, as well as in the diagnosis,treatment, and/or prevention of congenital uterine abnormalities, suchas bicornuate uterus, septate uterus, simple unicomuate uterus,unicornuate uterus with a noncavitary rudimentary horn, unicornuateuterus with a non-communicating cavitary rudimentary horn, unicornuateuterus with a communicating cavitary horn, arcuate uterus, uterinedidelfus, and T-shaped uterus.

[0632] Ovarian diseases and/or disorders include anovulation, polycysticovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarianhypofunction, ovarian insensitivity to gonadotropins, ovarianoverproduction of androgens, right ovarian vein syndrome, amenorrhea,hirutism, and ovarian cancer (including, but not limited to, primary andsecondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinomaof the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinousadenocarcinoma, and Ovarian Krukenberg tumors).

[0633] Cervical diseases and/or disorders include cervicitis, chroniccervicitis, mucopurulent cervicitis, cervical dysplasia, cervicalpolyps, Nabothian cysts, cervical erosion, cervical incompetence, andcervical neoplasms (including, for example, cervical carcinoma, squamousmetaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, andcolumnar cell neoplasia).

[0634] Additionally, diseases and/or disorders of the reproductivesystem include disorders and/or diseases of pregnancy, includingmiscarriage and stillbirth, such as early abortion, late abortion,spontaneous abortion, induced abortion, therapeutic abortion, threatenedabortion, missed abortion, incomplete abortion, complete abortion,habitual abortion, missed abortion, and septic abortion; ectopicpregnancy, anemia, Rh incompatibility, vaginal bleeding duringpregnancy, gestational diabetes, intrauterine growth retardation,polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa,hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticariaof pregnancy. Additionally, the albumin fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay be used in the diagnosis, treatment, and/or prevention of diseasesthat can complicate pregnancy, including heart disease, heart failure,rheumatic heart disease, congenital heart disease, mitral valveprolapse, high blood pressure, anemia, kidney disease, infectiousdisease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectioushepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus,Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis,chronic active hepatitis, cirrhosis of the liver, primary biliarycirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis,myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis,ovarian cysts, gallbladder disorders, and obstruction of the intestine.

[0635] Complications associated with labor and parturition includepremature rupture of the membranes, pre-term labor, post-term pregnancy,postmaturity, labor that progresses too slowly, fetal distress (e.g.,abnormal heart rate (fetal or maternal), breathing problems, andabnormal fetal position), shoulder dystocia, prolapsed umbilical cord,amniotic fluid embolism, and aberrant uterine bleeding.

[0636] Further, diseases and/or disorders of the postdelivery period,including endometritis, myometritis, parametritis, peritonitis, pelvicthrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis,saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage,and inverted uterus.

[0637] Other disorders and/or diseases of the female reproductive systemthat may be diagnosed, treated, and/or prevented by the albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention include, for example, Turner's syndrome,pseudohermaphroditism, premenstrual syndrome, pelvic inflammatorydisease, pelvic congestion (vascular engorgement), frigidity,anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.

[0638] Infectious Disease

[0639] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention can be used to treator detect infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation and differentiationof B and/or T cells, infectious diseases may be treated. The immuneresponse may be increased by either enhancing an existing immuneresponse, or by initiating a new immune response. Alternatively, fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention may also directly inhibit the infectiousagent, without necessarily eliciting an immune response.

[0640] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by albumin fusionproteins of the invention and/or polynucleotides encoding albumin fusionproteins of the invention. Examples of viruses, include, but are notlimited to Examples of viruses, include, but are not limited to thefollowing DNA and RNA viruses and viral families: Arbovirus,Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such asSmallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae(HTLV-I, HTLV-11, Lentivirus), and Togaviridae (e.g., Rubivirus).Viruses falling within these families can cause a variety of diseases orsymptoms, including, but not limited to: arthritis, bronchiollitis,respiratory syncytial virus, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunisticinfections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the commoncold, Polio, leukemia, Rubella, sexually transmitted diseases, skindiseases (e.g., Kaposi's, warts), and viremia. Albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention, can be used to treat or detect any of these symptomsor diseases. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g.,hepatitis B). In an additional specific embodiment fusion proteins ofthe invention and/or polynucleotides encoding albumin fusion proteins ofthe invention are used to treat patients nonresponsive to one or moreother commercially available hepatitis vaccines. In a further specificembodiment fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention are used to treatAIDS.

[0641] Similarly, bacterial and fungal agents that can cause disease orsymptoms and that can be treated or detected by albumin fusion proteinsof the invention and/or polynucleotides encoding albumin fusion proteinsof the invention include, but not limited to, the followingGram-Negative and Gram-positive bacteria, bacterial families, and fungi:Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans,Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g.,Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g.,Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia,Clostridium (e.g., Clostridium botulinum, Clostridium dificile,Clostridium perfringens, Clostridium tetani), Coccidioides,Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus,Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi,Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia,Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza typeB), Helicobacter, Legionella (e.g., Legionella pneumophia), Leptospira,Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium(e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio(e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea,Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g.,Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponemaspp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus(e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus andStreptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and CStreptococci), and Ureaplasmas. These bacterial, parasitic, and fungalfamilies can cause diseases or symptoms, including, but not limited to:antibiotic-resistant infections, bacteremia, endocarditis, septicemia,eye infections (e.g., conjunctivitis), uveitis, tuberculosis,gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDSrelated infections), paronychia, prosthesis-related infections, dentalcaries, Reiter's Disease, respiratory tract infections, such as WhoopingCough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,paratyphoid fever, food poisoning, Legionella disease, chronic and acuteinflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea,meningitis (e.g., mengitis types A and B), chlamydia, syphillis,diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneousabortions, birth defects, pneumonia, lung infections, ear infections,deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea,Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatorydiseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism,gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexuallytransmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),toxemia, urinary tract infections, wound infections, noscomialinfections. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used to treat or detect any of these symptoms or diseases. Inspecific embodiments, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention areused to treat: tetanus, diptheria, botulism, and/or meningitis type B.

[0642] Moreover, parasitic agents causing disease or symptoms that canbe treated, prevented, and/or diagnosed by fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention include, but not limited to, the following families or class:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis,Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, andTrichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodiumfalciparium, Plasmodium malariae and Plasmodium ovale). These parasitescan cause a variety of diseases or symptoms, including, but not limitedto: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g.,dysentery, giardiasis), liver disease, lung disease, opportunisticinfections (e.g., AIDS related), malaria, pregnancy complications, andtoxoplasmosis. Albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention, canbe used to treat, prevent, and/or diagnose any of these symptoms ordiseases. In specific embodiments, fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionare used to treat, prevent, and/or diagnose malaria.

[0643] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention could either be byadministering an effective amount of an albumin fusion protein of theinvnetion to the patient, or by removing cells from the patient,supplying the cells with a polynucleotide of the present invention, andreturning the engineered cells to the patient (ex vivo therapy).Moreover, the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention can beused as an antigen in a vaccine to raise an immune response againstinfectious disease.

[0644] Regeneration

[0645] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention can be used todifferentiate, proliferate, and attract cells, leading to theregeneration of tissues. (See, Science 276:59-87 (1997)). Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[0646] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0647] Moreover, fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may increaseregeneration of tissues difficult to heal. For example, increasedtendon/ligament regeneration would quicken recovery time after damage.Albumin fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

[0648] Similarly, nerve and brain tissue could also be regenerated byusing fusion proteins of the invention and/or polynucleotides encodingalbumin fusion proteins of the invention, to proliferate anddifferentiate nerve cells. Diseases that could be treated using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic disorders (e.g., spinal corddisorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated using the albumin fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention.

[0649] Gastrointestinal Disorders

[0650] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention, may be used to treat,prevent, diagnose, and/or prognose gastrointestinal disorders, includinginflammatory diseases and/or conditions, infections, cancers (e.g.,intestinal neoplasms (carcinoid tumor of the small intestine,non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)),and ulcers, such as peptic ulcers.

[0651] Gastrointestinal disorders include dysphagia, odynophagia,inflammation of the esophagus, peptic esophagitis, gastric reflux,submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas,lipomas, epidermal cancers, adeoncarcinomas, gastric retentiondisorders, gastroenteritis, gastric atrophy, gastric/stomach cancers,polyps of the stomach, autoimmune disorders such as pernicious anemia,pyloric stenosis, gastritis (bacterial, viral, eosinophilic,stress-induced, chronic erosive, atrophic, plasma cell, andMenetrier's), and peritoneal diseases (e.g., chyloperioneum,hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesentericvascular occlusion, panniculitis, neoplasms, peritonitis,pneumoperitoneum, bubphrenic abscess,).

[0652] Gastrointestinal disorders also include disorders associated withthe small intestine, such as malabsorption syndromes, distension,irritable bowel syndrome, sugar intolerance, celiac disease, duodenalulcers, duodenitis, tropical sprue, Whipple's disease, intestinallymphangiectasia, Crohn's disease, appendicitis, obstructions of theileum, Meckel's diverticulum, multiple diverticula, failure of completerotation of the small and large intestine, lymphoma, and bacterial andparasitic diseases (such as Traveler's diarrhea, typhoid andparatyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides),Hookworms (Ancylostoma duodenale), Threadworms (Enterobiusvermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus,Diphyllobothrium spp., and T. solium).

[0653] Liver diseases and/or disorders include intrahepatic cholestasis(alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholicfatty liver, reye syndrome), hepatic vein thrombosis, hepatolentriculardegeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenalsyndrome, portal hypertension (esophageal and gastric varices), liverabscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary andexperimental), alcoholic liver diseases (fatty liver, hepatitis,cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebicliver abscess), jaundice (hemolytic, hepatocellular, and cholestatic),cholestasis, portal hypertension, liver enlargement, ascites, hepatitis(alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune,hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis,viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitisD, hepatitis E), Wilson's disease, granulomatous hepatitis, secondarybiliary cirrhosis, hepatic encephalopathy, portal hypertension, varices,hepatic encephalopathy, primary biliary cirrhosis, primary sclerosingcholangitis, hepatocellular adenoma, hemangiomas, bile stones, liverfailure (hepatic encephalopathy, acute liver failure), and liverneoplasms (angiomyolipoma, calcified liver metastases, cystic livermetastases, epithelial tumors, fibrolamellar hepatocarcinoma, focalnodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma,hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liverhemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors ofliver, nodular regenerative hyperplasia, benign liver tumors (Hepaticcysts [Simple cysts, Polycystic liver disease, Hepatobiliarycystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymalhamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors[Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma),Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerativehyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi'ssarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosishepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittentporphyria, porphyria cutanea tarda), Zellweger syndrome).

[0654] Pancreatic diseases and/or disorders include acute pancreatitis,chronic pancreatitis (acute necrotizing pancreatitis, alcoholicpancreatitis), neoplasms (adenocarcinoma of the pancreas,cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cysticneoplasms, islet-cell tumors, pancreoblastoma), and other pancreaticdiseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreaticfistula, insufficiency)).

[0655] Gallbladder diseases include gallstones (cholelithiasis andcholedocholithiasis), postcholecystectomy syndrome, diverticulosis ofthe gallbladder, acute cholecystitis, chronic cholecystitis, bile ducttumors, and mucocele.

[0656] Diseases and/or disorders of the large intestine includeantibiotic-associated colitis, diverticulitis, ulcerative colitis,acquired megacolon, abscesses, fungal and bacterial infections,anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps(e.g., villous adenoma), colon carcinoma, colorectal cancer], colonicdiverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease,toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery),duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenalulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ilealdiseases (ileal neoplasms, ileitis), immunoproliferative smallintestinal disease, inflammatory bowel disease (ulcerative colitis,Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis,balantidiasis, blastocystis infections, cryptosporidiosis,dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula(rectal fistula), intestinal neoplasms (cecal neoplasms, colonicneoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps,jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferentloop syndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction [cecal volvulus], intussusception), intestinalperforation, intestinal polyps (colonic polyps, gardner syndrome,peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms),malabsorption syndromes (blind loop syndrome, celiac disease, lactoseintolerance, short bowl syndrome, tropical sprue, whipple's disease),mesenteric vascular occlusion, pneumatosis cystoides intestinalis,protein-losing enteropathies (intestinal lymphagiectasis), rectaldiseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenalulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer,Zollinger-Ellison syndrome), postgastrectomy syndromes (dumpingsyndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux(bile reflux), gastric antral vascular ectasia, gastric fistula, gastricoutlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis,stomach dilatation, stomach diverticulum, stomach neoplasms (gastriccancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastricpolyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,postoperative nausea and vomiting) and hemorrhagic colitis.

[0657] Further diseases and/or disorders of the gastrointestinal systeminclude biliary tract diseases, such as, gastroschisis, fistula (e.g.,biliary fistula, esophageal fistula, gastric fistula, intestinalfistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms,esophageal neoplasms, such as adenocarcinoma of the esophagus,esophageal squamous cell carcinoma, gastrointestinal neoplasms,pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinouscystic neoplasm of the pancreas, pancreatic cystic neoplasms,pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g.,bullous diseases, candidiasis, glycogenic acanthosis, ulceration,barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker'sdiverticulum), fistula (e.g., tracheoesophageal fistula), motilitydisorders (e.g., CREST syndrome, deglutition disorders, achalasia,spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaavesyndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatichernia (e.g., hiatal hernia); gastrointestinal diseases, such as,gastroenteritis (e.g., cholera morbus, norwalk virus infection),hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomachneoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma,stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoralhernia, inguinal hernia, obturator hernia, umbilical hernia, ventralhernia), and intestinal diseases (e.g., cecal diseases (appendicitis,cecal neoplasms)).

[0658] Chemotaxis

[0659] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may have chemotaxisactivity. A chemotaxic molecule attracts or mobilizes cells (e.g.,monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,epithelial and/or endothelial cells) to a particular site in the body,such as inflammation, infection, or site of hyperproliferation. Themobilized cells can then fight off and/or heal the particular trauma orabnormality.

[0660] Albumin fusion proteins of the invention and/or polynucleotidesencoding albumin fusion proteins of the invention may increasechemotaxic activity of particular cells. These chemotactic molecules canthen be used to treat inflammation, infection, hyperproliferativedisorders, or any immune system disorder by increasing the number ofcells targeted to a particular location in the body. For example,chemotaxic molecules can be used to treat wounds and other trauma totissues by attracting immune cells to the injured location. Chemotacticmolecules of the present invention can also attract fibroblasts, whichcan be used to treat wounds.

[0661] It is also contemplated that fusion proteins of the inventionand/or polynucleotides encoding albumin fusion proteins of the inventionmay inhibit chemotactic activity. These molecules could also be used totreat disorders. Thus, fusion proteins of the invention and/orpolynucleotides encoding albumin fusion proteins of the invention couldbe used as an inhibitor of chemotaxis.

[0662] Binding Activity

[0663] Albumin fusion proteins of the invention may be used to screenfor molecules that bind to the Therapeutic protein portion of the fusionprotein or for molecules to which the Therapeutic protein portion of thefusion protein binds. The binding of the fusion protein and the moleculemay activate (agonist), increase, inhibit (antagonist), or decreaseactivity of the fusion protein or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

[0664] Preferably, the molecule is closely related to the natural ligandof the Therapeutic protein portion of the fusion protein of theinvention, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, themolecule can be closely related to the natural receptor to which theTherapeutic protein portion of an albumin fusion protein of theinvention binds, or at least, a fragment of the receptor capable ofbeing bound by the Therapeutic protein portion of an albumin fusionprotein of the invention (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

[0665] Preferably, the screening for these molecules involves producingappropriate cells which express the albumin fusion proteins of theinvention. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli.

[0666] The assay may simply test binding of a candidate compound to analbumin fusion protein of the invention, wherein binding is detected bya label, or in an assay involving competition with a labeled competitor.Further, the assay may test whether the candidate compound results in asignal generated by binding to the fusion protein.

[0667] Alternatively, the assay can be carried out using cell-freepreparations, fusion protein/molecule affixed to a solid support,chemical libraries, or natural product mixtures. The assay may alsosimply comprise the steps of mixing a candidate compound with a solutioncontaining an albumin fusion protein, measuring fusion protein/moleculeactivity or binding, and comparing the fusion protein/molecule activityor binding to a standard.

[0668] Preferably, an ELISA assay can measure fusion protein level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure fusion protein level oractivity by either binding, directly or indirectly, to the albuminfusion protein or by competing with the albumin fusion protein for asubstrate.

[0669] Additionally, the receptor to which a Therapeutic protein portionof an albumin fusion protein of the invention binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, in cases wherein the Therapeuticprotein portion of the fusion protein corresponds to FGF, expressioncloning may be employed wherein polyadenylated RNA is prepared from acell responsive to the albumin fusion protein, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the albumin fusion protein. Transfected cellswhich are grown on glass slides are exposed to the albumin fusionprotein of the present invention, after they have been labeled. Thealbumin fusion proteins can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase.

[0670] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0671] As an alternative approach for receptor identification, a labeledalbumin fusion protein can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule for theTherapeutoc protein component of an albumin fusion protein of theinvention, the linked material may be resolved by PAGE analysis andexposed to X-ray film. The labeled complex containing the receptors ofthe fusion protein can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the genesencoding the putative receptors.

[0672] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of the fusionprotein, and/or Therapeutic protein portion or albumin component of analbumin fusion protein of the present invention, thereby effectivelygenerating agonists and antagonists of an albumin fusion protein of thepresent invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr.Opinion Biotechnol. 8:72-33 (1997); Harayama, S. Trends Biotechnol.16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76(1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13(1998); each of these patents and publications are hereby incorporatedby reference). In one embodiment, alteration of polynucleotides encodingalbumin fusion proteins of the invention and thus, the albumin fusionproteins encoded thereby, may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments into adesired molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides encoding albumin fusion proteins ofthe invention and thus, the albumin fusion proteins encoded thereby, maybe altered by being subjected to random mutagenesis by error-prone PCR,random nucleotide insertion or other methods prior to recombination. Inanother embodiment, one or more components, motifs, sections, parts,domains fragments, etc., of an albumin fusion protein of the presentinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0673] Other preferred fragments are biologically active fragments ofthe Therapeutic protein portion and/or albumin component of the albuminfusion proteins of the present invention. Biologically active fragmentsare those exhibiting activity similar, but not necessarily identical, toan activity of a Therapeutic protein portion and/or albumin component ofthe albumin fusion proteins of the present invention. The biologicalactivity of the fragments may include an improved desired activity, or adecreased undesirable activity.

[0674] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of an albuminfusion protein of the present invention. An example of such an assaycomprises combining a mammalian fibroblast cell, an albumin fusionprotein of the present invention, and the compound to be screened and³[H] thymidine under cell culture conditions where the fibroblast cellwould normally proliferate. A control assay may be performed in theabsence of the compound to be screened and compared to the amount offibroblast proliferation in the presence of the compound to determine ifthe compound stimulates proliferation by determining the uptake of ³[H]thymidine in each case. The amount of fibroblast cell proliferation ismeasured by liquid scintillation chromatography which measures theincorporation of ³[H] thymidine. Both agonist and antagonist compoundsmay be identified by this procedure.

[0675] In another method, a mammalian cell or membrane preparationexpressing a receptor for the Therapeutic protien component of a fusionprotine of the invention is incubated with a labeled fusion protein ofthe present invention in the presence of the compound. The ability ofthe compound to enhance or block this interaction could then bemeasured. Alternatively, the response of a known second messenger systemfollowing interaction of a compound to be screened and the receptor ismeasured and the ability of the compound to bind to the receptor andelicit a second messenger response is measured to determine if thecompound is a potential fusion protein. Such second messenger systemsinclude but are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

[0676] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the fusionprotein/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the albumin fusion proteins of theinvention from suitably manipulated cells or tissues.

[0677] Therefore, the invention includes a method of identifyingcompounds which bind to an albumin fusion protein of the inventioncomprising the steps of: (a) incubating a candidate binding compoundwith an albumin fusion protein of the present invention; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with an albumin fusion protein of thepresent invention, (b) assaying a biological activity, and (b)determining if a biological activity of the fusion protein has beenaltered.

[0678] Targeted Delivery

[0679] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for acomponent of an albumin fusion protein of the invention.

[0680] As discussed herein, fusion proteins of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering fusion proteins of the invention (including antibodies)that are associated with heterologous polypeptides or nucleic acids. Inone example, the invention provides a method for delivering aTherapeutic protein into the targeted cell. In another example, theinvention provides a method for delivering a single stranded nucleicacid (e.g., antisense or ribozymes) or double stranded nucleic acid(e.g., DNA that can integrate into the cell's genome or replicateepisomally and that can be transcribed) into the targeted cell.

[0681] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering an albumin fusion protein of the invention (e.g.,polypeptides of the invention or antibodies of the invention) inassociation with toxins or cytotoxic prodrugs.

[0682] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[0683] Drug Screening

[0684] Further contemplated is the use of the albumin fusion proteins ofthe present invention, or the polynucleotides encoding these fusionproteins, to screen for molecules which modify the activities of thealbumin fusion protein of the present invention or proteinscorresponding to the Therapeutic protein portion of the albumin fusionprotein. Such a method would include contacting the fusion protein witha selected compound(s) suspected of having antagonist or agonistactivity, and assaying the activity of the fusion protein followingbinding.

[0685] This invention is particularly useful for screening therapeuticcompounds by using the albumin fusion proteins of the present invention,or binding fragments thereof, in any of a variety of drug screeningtechniques. The albumin fusion protein employed in such a test may beaffixed to a solid support, expressed on a cell surface, free insolution, or located intracellularly. One method of drug screeningutilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the albumin fusionprotein. Drugs are screened against such transformed cells orsupernatants obtained from culturing such cells, in competitive bindingassays. One may measure, for example, the formulation of complexesbetween the agent being tested and an albumin fusion protein of thepresent invention.

[0686] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thealbumin fusion proteins of the present invention. These methods comprisecontacting such an agent with an albumin fusion protein of the presentinvention or a fragment thereof and assaying for the presence of acomplex between the agent and the albumin fusion protein or a fragmentthereof, by methods well known in the art. In such a competitive bindingassay, the agents to screen are typically labeled. Following incubation,free agent is separated from that present in bound form, and the amountof free or uncomplexed label is a measure of the ability of a particularagent to bind to the albumin fusion protein of the present invention.

[0687] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to an albuminfusion protein of the present invention, and is described in greatdetail in European Patent Application 84/03564, published on Sep. 13,1984, which is incorporated herein by reference herein. Briefly stated,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with an albumin fusion protein of thepresent invention and washed. Bound peptides are then detected bymethods well known in the art. Purified albumin fusion protein may becoated directly onto plates for use in the aforementioned drug screeningtechniques. In addition, non-neutralizing antibodies may be used tocapture the peptide and immobilize it on the solid support.

[0688] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding analbumin fusion protein of the present invention specifically competewith a test compound for binding to the albumin fusion protein orfragments thereof. In this manner, the antibodies are used to detect thepresence of any peptide which shares one or more antigenic epitopes withan albumin fusion protein of the invention.

[0689] Binding Peptides and Other Molecules

[0690] The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind albumin fusion proteins ofthe invention, and the binding molecules identified thereby. Thesebinding molecules are useful, for example, as agonists and antagonistsof the albumin fusion proteins of the invention. Such agonists andantagonists can be used, in accordance with the invention, in thetherapeutic embodiments described in detail, below.

[0691] This method comprises the steps of:

[0692] contacting an albumin fusion protein of the invention with aplurality of molecules; and

[0693] identifying a molecule that binds the albumin fusion protein.

[0694] The step of contacting the albumin fusion protein of theinvention with the plurality of molecules may be effected in a number ofways. For example, one may contemplate immobilizing the albumin fusionprotein on a solid support and bringing a solution of the plurality ofmolecules in contact with the immobilized polypeptides. Such a procedurewould be akin to an affinity chromatographic process, with the affinitymatrix being comprised of the immobilized albumin fusion protein of theinvention. The molecules having a selective affinity for the albuminfusion protein can then be purified by affinity selection. The nature ofthe solid support, process for attachment of the albumin fusion proteinto the solid support, solvent, and conditions of the affinity isolationor selection are largely conventional and well known to those ofordinary skill in the art.

[0695] Alternatively, one may also separate a plurality of polypeptidesinto substantially separate fractions comprising a subset of orindividual polypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by an albumin fusion protein of the invention, optionally inthe presence of an inducer should one be required for expression, todetermine if any selective affinity interaction takes place between thealbumin fusion protein and the individual clone. Prior to contacting thealbumin fusion protein with each fraction comprising individualpolypeptides, the polypeptides could first be transferred to a solidsupport for additional convenience. Such a solid support may simply be apiece of filter membrane, such as one made of nitrocellulose or nylon.In this manner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for analbumin fusion protein of the invention. Furthermore, the amino acidsequence of the polypeptide having a selective affinity for an albuminfusion protein of the invention can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

[0696] In certain situations, it may be desirable to wash away anyunbound polypeptides from a mixture of an albumin fusion protein of theinvention and the plurality of polypeptides prior to attempting todetermine or to detect the presence of a selective affinity interaction.Such a wash step may be particularly desirable when the albumin fusionprotein of the invention or the plurality of polypeptides are bound to asolid support.

[0697] The plurality of molecules provided according to this method maybe provided by way of diversity libraries, such as random orcombinatorial peptide or nonpeptide libraries which can be screened formolecules that specifically bind an albumin fusion protein of theinvention. Many libraries are known in the art that can be used, e.g.,chemically synthesized libraries, recombinant (e.g., phage displaylibraries), and in vitro translation-based libraries. Examples ofchemically synthesized libraries are described in Fodor et al., Science251:767-773 (1991); Houghten et al., Nature 354:84-86 (1991); Lam etal., Nature 354:82-84 (1991); Medynski, Bio/Technology 12:709-710(1994); Gallop et al., J. Medicinal Chemistry 37(9):1233-1251 (1994);Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993); Erbet al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houghten etal., Biotechniques 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad.Sci. USA 91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA90:11708-11712 (1993); PCT Publication No. WO 93/20242; and Brenner andLerner, Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992).

[0698] Examples of phage display libraries are described in Scott etal., Science 249:386-390 (1990); Devlin et al., Science, 249:404-406(1990); Christian et al., 1992, J. Mol. Biol. 227:711-718 1992);Lenstra, J. Immunol. Meth. 152:149-157 (1992); Kay et al., Gene128:59-65 (1993); and PCT Publication No. WO 94/18318 dated Aug. 18,1994.

[0699] In vitro translation-based libraries include but are not limitedto those described in PCT Publication No. WO 91!05058 dated Apr. 18,1991; and Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026(1994).

[0700] By way of examples of nonpeptide libraries, a benzodiazepinelibrary (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712(1994)) can be adapted for use. Peptoid libraries (Simon et al., Proc.Natl. Acad. Sci. USA 89:9367-9371 (1992)) can also be used. Anotherexample of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal. (Proc. Natl. Acad. Sci. USA 91:11138-11142 (1994)).

[0701] The variety of non-peptide libraries that are useful in thepresent invention is great. For example, Ecker and Crooke(Bio/Technology 13:351-360 (1995) list benzodiazepines, hydantoins,piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,aminimides, and oxazolones as among the chemical species that form thebasis of various libraries.

[0702] Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

[0703] Non-peptide oligomer libraries utilize a large number of monomersthat are assembled together in ways that create new shapes that dependon the order of the monomers. Among the monomer units that have beenused are carbamates, pyrrolinones, and morpholinos. Peptoids,peptide-like oligomers in which the side chain is attached to the alphaamino group rather than the alpha carbon, form the basis of anotherversion of non-peptide oliaomer libraries. The first non-peptideotigomer libraries utilized a single type of monomer and thus containeda repeating backbone. Recent libraries have utilized more than onemonomer, giving the libraries added flexibility.

[0704] Screening the libraries can be accomplished by any of a varietyof commonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley et al., Adv. Exp. Med.Biol. 251:215-218 (1989); Scott et al,. Science 249:386-390 (1990);Fowlkes et al., BioTechniques 13:422-427 (1992); Oldenburg et al., Proc.Natl. Acad. Sci. USA 89:5393-5397 (1992); Yu et al., Cell 76:933-945(1994); Staudt et al., Science 241:577-580 (1988); Bock et al., Nature355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci. USA89:6988-6992 (1992); Ellington et al., Nature 355:850-852 (1992); U.S.Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No.5,198,346, all to Ladner et al.; Rebar et al., Science 263:671-673(1993); and PCT Publication No. WO 94/18318.

[0705] In a specific embodiment, screening to identify a molecule thatbinds an albumin fusion protein of the invention can be carried out bycontacting the library members with an albumin fusion protein of theinvention immobilized on a solid phase and harvesting those librarymembers that bind to the albumin fusion protein. Examples of suchscreening methods, termed “panning” techniques are described by way ofexample in Parmley et al., Gene 73:305-318 (1988); Fowlkes et al.,BioTechniques 13:422-427 (1992); PCT Publication No. WO 94/18318; and inreferences cited herein.

[0706] In another embodiment, the two-hybrid system for selectinginteracting proteins in yeast (Fields et al., Nature 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991) can be usedto identify molecules that specifically bind to polypeptides of theinvention.

[0707] Where the binding molecule is a polypeptide, the polypeptide canbe conveniently selected from any peptide library, including randompeptide libraries, combinatorial peptide libraries, or biased peptidelibraries. The term “biased” is used herein to mean that the method ofgenerating the library is manipulated so as to restrict one or moreparameters that govern the diversity of the resulting collection ofmolecules, in this case peptides.

[0708] Thus, a truly random peptide library would generate a collectionof peptides in which the probability of finding a particular amino acidat a given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

[0709] As mentioned above, in the case of a binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a binding polypeptide has in the range of 15-100 aminoacids, or 20-50 amino acids.

[0710] The selected binding polypeptide can be obtained by chemicalsynthesis or recombinant expression.

[0711] Other Activities

[0712] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention, may be employed intreatment for stimulating re-vascularization of ischemic tissues due tovarious disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. The albumin fusion proteins of theinvention and/or polynucleotides encoding albumin fusion proteins of theinvention may also be employed to stimulate angiogenesis and limbregeneration, as discussed above.

[0713] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedfor treating wounds due to injuries, burns, post-operative tissuerepair, and ulcers since they are mitogenic to various cells ofdifferent origins, such as fibroblast cells and skeletal muscle cells,and therefore, facilitate the repair or replacement of damaged ordiseased tissue.

[0714] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. An albumin fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention mayhave the ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

[0715] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may be also beemployed to prevent skin aging due to sunburn by stimulatingkeratinocyte growth.

[0716] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedfor preventing hair loss, since FGF family members activate hair-formingcells and promotes melanocyte growth. Along the same lines, an albuminfusion protein of the invention and/or polynucleotide encoding analbumin fusion protein of the invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellswhen used in combination with other cytokines.

[0717] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be employedto maintain organs before transplantation or for supporting cell cultureof primary tissues. An albumin fusion protein of the invention and/orpolynucleotide encoding an albumin fusion protein of the invention mayalso be employed for inducing tissue of mesodermal origin todifferentiate in early embryos.

[0718] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also increase ordecrease the differentiation or proliferation of embryonic stem cells,besides, as discussed above, hematopoietic lineage.

[0719] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be used tomodulate mammalian characteristics, such as body height, weight, haircolor, eye color, skin, percentage of adipose tissue, pigmentation,size, and shape (e.g., cosmetic surgery). Similarly, an albumin fusionprotein of the invention and/or polynucleotide encoding an albuminfusion protein of the invention may be used to modulate mammalianmetabolism affecting catabolism, anabolism, processing, utilization, andstorage of energy.

[0720] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may be used tochange a mammal's mental state or physical state by influencingbiorhythms, caricadic rhythms, depression (including depressivedisorders), tendency for violence, tolerance for pain, reproductivecapabilities (preferably by Activin or Inhibin-like activity), hormonalor endocrine levels, appetite, libido, memory, stress, or othercognitive qualities.

[0721] An albumin fusion protein of the invention and/or polynucleotideencoding an albumin fusion protein of the invention may also be used asa food additive or preservative, such as to increase or decrease storagecapabilities, fat content, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional components.

[0722] The above-recited applications have uses in a wide variety ofhosts. Such hosts include, but are not limited to, human, murine,rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig,micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, andhuman. In specific embodiments, the host is a mouse, rabbit, goat,guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferredembodiments, the host is a mammal. In most preferred embodiments, thehost is a human.

[0723] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0724] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the alterations detected in thepresent invention and practice the claimed methods. The followingworking examples therefore, specifically point out preferred embodimentsof the present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

EXAMPLES Example 1 Preparation of HA-hGH Fusion Proteins

[0725] An HA-hGH fusion protein was prepared as follows:

[0726] Cloning of hGH cDNA

[0727] The hGH cDNA was obtained from a human pituitary gland cDNAlibrary (catalogue number HL1097v, Clontech Laboratories, Inc) by PCRamplification. Two oligonucleotides suitable for PCR amplification ofthe hGH cDNA, HGHI and HGH2, were synthesized using an AppliedBiosystems 380B Oligonucleotide Synthesizer. (SEQ ID NO: 1) HGH1:5′-CCCAAGAATTCCCTTATCCAGGC-3′ (SEQ ID NO: 2) HGH2:5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′

[0728] HGH 1 and HGH2 differed from the equivalent portion of the hGHcDNA sequence (Martial et. al., 1979) by two and three nucleotides,respectively, such that after PCR amplification an EcoRI site would beintroduced to the 5′ end of the cDNA and a BamHI site would beintroduced into the 3′ end of the cDNA. In addition, HGH2 contained aHindIII site immediately downstream of the hGH sequence.

[0729] PCR amplification using a Perkin-Elmer-Cetus Thermal Cycler 9600and a Perkin-Elmer-Cetus PCR kit, was performed using single-strandedDNA template isolated from the phage particles of the cDNA library asfollows: 10 μL phage particles were lysed by the addition of 10 μL phagelysis buffer (280 μg/mL proteinase K in TE buffer) and incubation at 55°C. for 15 min followed by 85° C. for 15 min. After a 1 min. incubationon ice, phage debris was pelleted by centrifugation at 14,000 rpm for 3min. The PCR mixture contained 6 μL of this DNA template, 0.1 μM of eachprimer and 200 μM of each deoxyribonucleotide. PCR was carried out for30 cycles, denaturing at 94° C. for 30 s, annealing at 65° C. for 30 sand extending at 72° C. for 30 s, increasing the extension time by 1 sper cycle.

[0730] Analysis of the reaction by gel electrophoresis showed a singleproduct of the expected size (589 base pairs).

[0731] The PCR product was purified using Wizard PCR Preps DNAPurification System (Promega Corp) and then digested with EcoRI andHindIII. After further purification of the EcoRI-HindIII fragment by gelelectrophoresis, the product was cloned into pUC19 (GIBCO BRL) digestedwith EcoRI and HindIII, to give pHGH1. DNA sequencing of the EcoRIHindIII region showed that the PCR product was identical in sequence tothe hGH sequence (Martial et al., 1979), except at the 5′ and 3′ ends,where the EcoRI and BamHI sites had been introduced, respectively.

[0732] Expression of the hGH cDNA.

[0733] The polylinker sequence of the phagemid pBluescribe (+)(Stratagene) was replaced by inserting an oligonucleotide linker, formedby annealing two 75-mer oligonucleotides, between the EcoRI and HindIIIsites to form pBST(+). The new polylinker included a unique NotI site.

[0734] The NotI HA expression cassette of pAYE309 (EP 431 880)comprising the PRBI promoter, DNA encoding the HA/MFα-1 hybrid leadersequence, DNA encoding HA and the ADH1 terminator, was transferred topBST(+) to form pHA1. The HA coding sequence was removed from thisplasmid by digestion with HindIII followed by religation to form pHA2.

[0735] Cloning of the hGH cDNA, as described in Example 1, provided thehGH coding region lacking the pro-hGH sequence and the first 8 basepairs (bp) of the mature hGH sequence. In order to construct anexpression plasmid for secretion of hGH from yeast, a yeast promoter,signal peptide and the first 8 bp of the hGH sequence were attached tothe 5′ end of the cloned hGH sequence as follows: The HindIII-SfaNIfragment from pHA 1 was attached to the 5′ end of the EcoRIHindIIIfragment from pHGHI via two synthetic oligonucleotides, HGH3 and HGH4(which can anneal to one another in such a way as to generate a doublestranded fragment of DNA with sticky ends that can anneal with SfaNlandEcoRI sticky ends): HGH3: 5′-GATAAAGATTCCCAAC-3′ (SEQ ID NO: 3) HGH4:5′-AATTGTTGGGAATCTTT-3′ (SEQ ID NO: 4)

[0736] The HindIII fragment so formed was cloned into HindIII-digestedpHA2 to make pHGH2, such that the hGH cDNA was positioned downstream ofthe PRBI promoter and HA/MFα-1 fusion leader sequence (see,International Publication No. WO 90/01063). The NotI expression cassettecontained in pHGH2, which included the ADH1 terminator downstream of thehGH cDNA, was cloned into NotI-digested pSAC35 (Sleep et al.,BioTechnology 8:42 (1990)) to make pHGH12. This plasmid comprised theentire 2 μm plasmid to provide replication functions and the LEU2 genefor selection of transformants.

[0737] pHGH12 was introduced into S. cerevisiae D88 by transformationand individual transformants were grown for 3 days at 30° C. in 10 mLYEPD (1% w/v yeast extract, 2% w/v, peptone, 2% w/v, dextrose).

[0738] After centrifugation of the cells, the supernatants were examinedby SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were found tocontain protein which was of the expected size and which was recognizedby anti-hGH antiserum (Sigma, Poole, UK) on Western blots.

[0739] Cloning and Expression of an HA-hGH Fusion Protein.

[0740] In order to fuse the HA cDNA to the 5′ end of the hGH cDNA, thepHA1 HindIII-Bsu361 fragment (containing most of the HA cDNA) was joinedto the pHGH1 EcoRI-HindIII fragment (containing most of the hGH cDNA)via two oligonucleotides, HGH7 and HGH8 HGH7: 5′- TTAGGCTTATTCCCAAC 3′(SEQ ID NO: 5) HGH8: 5′- AATTGTTGGGAATAAGCC 3′ (SEQ ID NO: 6)

[0741] The HindIII fragment so formed was cloned into pHA2 digested withHindIII to make pHGH10, and the NotI expression cassette of this plasmidwas cloned into NotI-digested pSAC35 to make pHGH16. pHGH16 was used totransform S. cerevisiae D88 and supernatants of cultures were analyzedas described above. A predominant band was observed that had a molecularweight of approximately 88 kD, corresponding to the combined masses ofHA and hGH. Western blotting using anti-HA and anti-hGH antisera (Sigma)confirmed the presence of the two constituent parts of the albuminfusion protein.

[0742] The albumin fusion protein was purified from culture supernatantby cation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected albuminsequence.

[0743] An in vitro growth hormone activity assay (Ealey et al., GrowthRegulation 5:36 (1995)) indicated that the albumin fusion proteinpossessed full hGH activity. In a hypophysectomised rat weight gainmodel, performed essentially as described in the European Pharmacopoeia(1987, monograph 556), the fusion molecule was more potent than hGH whenthe same number of units of activity (based on the above in vitro assay)were administered daily. Further experiments in which the albumin fusionprotein was administered once every four days showed a similar overallgrowth response to a daily administration of hGH. Pharmacokineticexperiments in which 125% labeled protein was administered to ratsindicated an approximately ten-fold increase in circulatory half-lifefor the albumin fusion protein compared to hGH.

[0744] A similar plasmid was constructed in which DNA encoding the S.cerevisiae invertase (SUC2) leader sequence replaced the sequence forthe hybrid leader, such that the encoded leader and the junction (↓)with the HA sequence were as follows:

[0745] . . . MLLQAFLFLLAGFAAKISA↓DAHKS . . . (SEQ ID NO:7) Invertaseleader HA sequence . . .

[0746] On introduction into S. cerevisiae DBI, this plasmid directed theexpression and secretion of the albumin fusion protein at a levelsimilar to that obtained with pHGH16. Analysis of the N-terminus of thealbumin fusion protein indicated precise and efficient cleavage of theleader sequence from the mature protein.

[0747] Cloning and Expression of an hGH-HA Fusion Protein.

[0748] In order to fuse the hGH cDNA to the 5′ end of the HA cDNA, theHA cDNA was first altered by site-directed mutagenesis to introduce anEcoNI site near the 5′ end of the coding region. This was done by themethod of Kunkel et al. (Methods in Enzymol. 154:367 (1987)) usingsingle-stranded DNA template prepared from pHAl and a syntheticoligonucleotide, LEU4:

[0749] LEU4: 5′-GAGATGCACACCTGAGTGAGG-3′ (SEQ ID NO:8)

[0750] Site-directed mutagenesis using this oligonucleotide changed thecoding sequence of the HA cDNA from Lys4 to Leu4 (K4L). However, thischange was repaired when the hGH cDNA was subsequently joined at the 5′end by linking the pHGH2 NotI-BamHI fragment to the EcoNI-NotI fragmentof the mutated pHAI, via the two oligonucleotides HGH5 and HGH6: (SEQ IDNO: 9) HGH5: 5′-GATCCTGTGGCTTCGATGCACACAAGA-3′ (SEQ ID NO: 10) HGH6:5′-CTCTTGTGTGCATCGAAGCCACAG-3′

[0751] The Notl fragment so formed was cloned into NotI-digested pSAC35to make pHGH14. pHGH14 was used to transform S. cerevisiae D88 andsupernatants of culture were analyzed as above. A predominant band wasobserved that had a molecular weight of approximately 88 kD,corresponding to the combined masses of hGH and HA. Western blottingusing anti-HA and anti-hGH antisera confirmed the presence of the twoconstituent parts of the albumin fusion protein.

[0752] The albumin fusion protein was purified from culture supernatantby cation exchange chromatography, followed by anion exchange and gelpermeation chromatography. Analysis of the N-terminus of the protein byamino acid sequencing confirmed the presence of the expected hGHsequence.

[0753] In vitro studies showed that the albumin fusion protein retainedhGH activity, but was significantly less potent than an albumin fusionprotein comprising full length HA (1-585) as the N-terminal portion andhGH as the C-terminal portion, as described above.

[0754] Construction of Plasmids for the Expression of hGH Fusions toDomains of HA.

[0755] Fusion polypeptides were made in which the hGH molecule was fusedto the first two domains of HA (residues 1 to 387). Fusion to the Nterminus of hGH was achieved by joining the pHA1 HindIII-Sapl fragment,which contained most of the coding sequence for domains 1 and 2 of HA,to the pHGHI EcoRI-HindIII fragment, via the oligonucleotides HGH 11 andHGH 12: (SEQ ID NO: 11) HGH11: 5′-TGTGGAAGAGCCTCAGAATTTATTCCCAAC-3′ (SEQID NO: 12) HGH12: 5′-AATTGTTGGGAATAAATTCTGAGGCTCTTCC-3′

[0756] The HindIII fragment so formed was cloned into HindIII-digestedpHA2 to make pHGH37 and the Notl expression cassette of this plasmid wascloned into Notl-digested pSAC35.

[0757] The resulting plasmid, pHGH38, contained an expression cassettethat was found to direct secretion of the fusion polypeptide into thesupernatant when transformed into S. cerevisiae DB 1. Western blottingusing anti-HA and anti-hGH antisera confirmed the presence of the twoconstituent parts of the albumin fusion protein.

[0758] The albumin fusion protein was purified from culture supernatantby cation exchange chromatography followed by gel permeationchromatography.

[0759] In vivo studies with purified protein indicated that thecirculatory half-life was longer than that of hGH, and similar to thatof an albumin fusion protein comprising full-length HA (1-585) as theN-terminal portion and hGH as the C-terminal portion, as describedabove. In vitro studies showed that the albumin fusion protein retainedhGH activity.

[0760] Using a similar strategy as detailed above, an albumin fusionprotein comprising the first domain of HA (residues 1-194) as theN-terminal portion and hGH as the C-terminal portion, was cloned andexpressed in S. cerevisiae DBL. Western blotting of culture supernatantusing anti-HA and anti-hGH antisera confirmed the presence of the twoconstituent parts of the albumin fusion protein.

[0761] Fusion of HA to hGH Using a Flexible Linker Sequence

[0762] Flexible linkers, comprising repeating units of[Gly-Gly-Gly-Gly-Ser]_(n), where n was either 2 or 3, were introducedbetween the HA and hGH albumin fusion protein by cloning of theoligonucleotides HGH16, HGH17, HGH18 and HGH19: HGH16:5′-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTTCCCA AC-3′ (SEQ ID NO: 13)HGH17: 5′-AATTGTTGGGAAAGATCCACCACCGCCGGATCCACCGCCACCTAAGCC-3′ (SEQ IDNO: 14) HGH18: 5′-TTAGGCTTAGGCGGTGGTGGATCTGGTGGCGGCGGATCTGGTGGCGGTGGATCC(SEQ ID NO: 15)        TTCCCAAC-3′ HGH19:5′-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCACCA (SEQ ID NO: 16)       GATCCACCACCGCCTAAGCC-3′

[0763] Annealing of HGH16 with HGH17 resulted in n=2, while HGH18annealed to HGH19 resulted in n=3. After annealing, the double-strandedoligonucleotides were cloned with the EcoRI-Bsu361 fragment isolatedfrom pHGH1 into Bsu361-digested pHGH10 to make pHGH56 (where n=2) andpHGH57 (where n=3). The NotI expression cassettes from these plasmidswere cloned into NotI-digested pSAC35 to make pHGH58 and pHGH59,respectively.

[0764] Cloning of the oligonucleotides to make pHGH56 and pHGH57introduced a BamHI site in the linker sequences. It was thereforepossible to construct linker sequences in which n=1 and n=4, by joiningeither the HindIII-BamHI fragment from pHGH56 to the BamHI-HindIIIfragment from pHGH57 (making n=1), or the HindIII-BamHI fragment frompHGH57 to the BamHI-HindIII fragment from pHGH56 (making n=2). Cloningof these fragments into the HindIII site of pHA2, resulted in pHGH60(n=1) and pHGH61 (n=4). The NotI expression cassettes from pHGH60 andpHGH61 were cloned into NotI-digested pSAC35 to make pHGH62 and pHGH63,respectively.

[0765] Transformation of S. cerevisiae with pHGH58, pHGH59, pHGH62 andpHGH63 resulted in transformants that secreted the fusion polypeptidesinto the supernatant. Western blotting using anti-HA and anti-hGHantisera confirmed the presence of the two constituent parts of thealbumin fusion proteins.

[0766] The albumin fusion proteins were purified from culturesupernatant by cation exchange chromatography, followed by anionexchange and gel permeation chromatography. Analysis of the N-termini ofthe proteins by amino acid sequencing confirmed the presence of theexpected albumin sequence. Analysis of the purified proteins byelectrospray mass spectrometry confirmed an increase in mass of 315 D(n=1), 630 D (n=2), 945 D (n=3) and 1260 D (n=4) compared to the HA-hGHfusion protein described above. The purified protein was found to beactive in vitro.

[0767] Increased Shelf-Life of HA-hGH Fusion Proteins: Methods

[0768] HA-hGH and hGH were separately diluted in cell culture mediacontaining 5% horse serum to final concentrations of 100-200 μg/ml andincubated at 4, 37 or 50° C. At time zero and at weekly intervalsthereafter, aliquots of the samples were tested for their biologicalactivity in the Nb2 cell proliferation assay, and the data normalized tothe biological activity of the control (hGH solution at time zero). Inother assays hGH and HA-hGH were incubated in phosphate buffer saline inat 4, 37 and 50 degree C.

[0769] Nb2 cell proliferation assay: The growth of these cells isdependent on hGH or other lactogenic hormones. In a typical experiment10⁴ cells/well are plated in 96-well plate in the presence of differentconcentration of hGH or HA-hGH in media such as DMEM containing 5-10%horse serum for 24-48 hrs in the incubator. After the incubation period,1: 10 volume of MTT (5 mg/ml in H₂O) is added to each well and the plateis incubated for a further 6-16 hrs. The growing cells convert MTT toinsoluble formazan. The formazan is solublized by acidic isopropanol,and the color produced is measured at 570 nm on microtiter plate reader.The extent of formazan formation reflects the level of cellularproliferation.

[0770] Increased Shelf-Life of HA-h1 GH Fusion Proteins: Results

[0771] The fusion of Therapeutic proteins to albumin confers stabilityin aqueous or other solution. FIG. 1 depicts the extended shelf-life ofan HA fusion protein in terms of the biological activity of HA-hGHremaining after storage in cell culture media for up to 5 weeks at 37°C. A solution of 200 μg/ml HA-hGH was prepared in tissue culture mediacontaining 5% horse serum, and the solution incubated at 37° C. startingat time zero. At the indicated times, a sample was removed and testedfor its biological activity in the Nb2 cell assay, at 2 ng/ml finalconcentration. As shown in FIG. 1, the biological activity of HA-hGHremains essentially intact (within experimental variation) after 5 weeksof incubation at 37° C. The recombinant hGH used as control for thisexperiment lost its biological activity in the first week of theexperiment.

[0772]FIG. 2 shows the stability of HA-hGH after storage in cell culturemedia for up to 3 weeks at 4, 37, or 50° C. At time zero, a solution ofHA-hGH was prepared in tissue culture media containing 5% horse serum,and incubated at 4, 37, and 50° C. At the indicated periods a sample wasremoved and assayed for its biological activity in the Nb2 cellproliferation assay, at 60 ng/ml final concentration. HA-hGH retainsover 90% of its initial activity at all temperatures tested for at least3 weeks after incubation while hGH loses its biological activity withinthe first week. This level of activity is further retained for at least7 weeks at 37° C. and 5 weeks at 50° C. These results indicate thatHA-hGH is highly stable in aqueous solution even under temperaturestress.

[0773]FIGS. 3A and 3B show the stable biological activity of HA-hGHcompared to hGH in the Nb2 cell proliferation assay. Nb2 cells weregrown in the presence of increasing concentrations of recombinant hGH orHA-hGH, added at time zero. The cells were incubated for 24 or 48 hoursbefore measuring the extent of proliferation by the MTT method. Theincreased stability of HA-hGH in the assay results in essentially thesame proliferative activity at 24 hours (FIG. 3A) as at 48 hours (FIG.3B) while hGH shows a significant reduction in its proliferativeactivity after 48 hours of incubation (FIGS. 3A and 3B). Compared tohGH, the HA-hGH has lower biological potency after 1 day; the albuminfusion protein is about 5 fold less potent than hGH. However, after 2days the HA-hGH shows essentially the same potency as hGH due to theshort life of hGH in the assay. This increase in the stability of thehGH as an albumin fusion protein has a major unexpected impact on thebiological activity of the protein. Although the potency of the albuminfusion proteins is slightly lower than the unfused counterparts in rapidbioassays, their biological stability results in much higher biologicalactivity in the longer term in vitro assay or in vivo assays.

Example 2 Preparation of HA-Fusion Proteins

[0774]FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as thebase vector for cloning the cDNAs of therapeutic partners to formHA-fusions. For example, digestion of this vector with the restrictionenzymes Bsu36I/Partial HindIII will allow for the insertion of a cDNAmodified at the 5′ end to encode the last 5 amino acids of HA includingthe Bsu36I site and at the 3′ end to include a double stop codon andHindIII site. As another example, digestion of this vector with therestriction enzymes Bsu361/, SphI allows for the insertion of a cDNAmodified at the 5′ end to encode the last 5 amino acids of HA includingthe Bsu36I site and at the 3′ end to include a double stop codon,HindIII site and the ADHI terminator sequence up to and including theSphI site.

[0775] This plasmid may easily be modified by one of skill in the art,for example, to modify, add or delete restriction sites so that one maymore easily clone a Therapeutic protein, or fragment or variant of intothe vector for the purpose of making an albumin fusion protein of theinvention.

[0776] For example, for the purpose of making an albumin fusion proteinwhere the Therapeutic moiety is placed N-terminal to the (mature)albumin protein, restriction sites were added at the 5′ end of the DNAencoding HA in pPPC0005 shown in FIG. 4).

[0777] Because it was desired to add unique XhoI and ClaI sites at the5′ end of the DNA encoding the HA protein in pPPC0005, it was firstnecessary to remove those same sites from the plasmid (located 3′ of theADH1 terminator sequence). This was accomplished by cutting pPPC0005with XhoI and ClaI, filling in the sticky ends with T4 DNA polymerase,and religating the blunt ends to create pPPC0006 Engineering the Xho andCla I restriction sites into the Fusion leader sequence just 5′ of theDNA encoding the HA protein in pPPC0006 was accomplished using tworounds of PCR. The first pair of oligonucleotides are those of SEQ IDNO:19 and SEQ ID NO:20.

[0778] SEQ ID 19 contains four point mutations relative to the DNAsequence encoding the Fusion leaadr sequence and the beginning of the HAprotein. These mutations are necessary to create the XhoI site in thefusion leader sequence and the Cla I site just at the beginning of theDNA encoding the HA protein. These four mutations are underlined in thesequence shown below.

[0779] In pPPC0006 the nucleotides at these four positions from 5′ to 3′are T, G, T, and G.5′-GCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAAGAT TTGGG-3′ (SEQID NO:19) 5′-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3′ (SEQ ID NO:20). A second round of PCR is then performed with anupstream flanking primer, 5′-TACAAACTTAAGAGTCCAATTAGC-3′ (SEQ ID NO:21)and a downstream flanking primer 5′-CACTTCTCTAGAGTGGTTTCATATGTCTT-3′(SEQ ID NO:22). The resulting PCR product is then purified and thendigested with AflI and XbaI and ligated into the same sites in pPPC0006creating pScCHSA. The resulting plasmid will have an XhoI sitesengineered into the fusion leader sequence. The presence of the XhoIsite creates a single amino acid change in the end of fusion leadersequence from LDKR to LEKR. The D to E change will not be present in thefinal albumin fusion protein expression plasmid if one ligates into theXhoI and Cla I sites a fragment comprising the Therapeutic moiety whichhas a 5′ SalI sticky end (which is compatible with the XhoI end) and a3′ ClaI end. Ligation of the XhoI to the SalI restores the originalamino acid sequence of the Fusion leader sequence. The Therapeuticprotein moiety may be inserted after the Kex2 site (Kex2 claeves afterthe dibasic amino acid sequence KR at the end of the Fusion leadersequence) and before the ClaI site.

[0780] In addition, for the purpose of making an albumin fusion proteinwhere the Therapeutic moiety is placed C-terminal to the (mature)albumin protein, four, eight-base-pair restriction sites were added atthe 3′ end of the DNA encoding HA in pScCHSA. As an example, it was feltto be desirable to incorporate AscI, FseI, and PmeI restriction sites inbetween the Bsu361 and HindIII sites at the end of the DNA encoding theHA protein in pScCHSA. This was accomplished through the use of twocomplementary synthetic oligonucleotides (SEQ ID NO:19 and SEQ ID NO:20)which contain the desired restriction sites.5′-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAACTAAGCT TAATTCT-3′ (SEQID NO:23) and 5-AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3′ (SEQ ID NO:24). These oligonucleotides may be annealed anddigested with Bsu36I and HindIII and ligated into the same sites locatedat the end of the DNA encoding the HA protein in pScCHSA creatingpScNHSA, using techniques known in the art.

[0781] Making Vectors Comprising Albumin Fusion Proteins where theAlbumin Moiety is N-Terminal to the Therapeutic Moiety.

[0782] The DNA encoding the Therapeutic moiety may be PCR amplifiedusing primers that will add DNA encoding, the last five amino acids ofthe HA (and containing the Bsu361 site) onto the 5′ end of the DNAencoding a Therapeutic protein and a STOP codon and appropriate cloningsites onto the 3′ end of the coding sequence. For instance, the forwardprimer used to amplify the DNA encoding a Therapeutic protein might havethe sequence, 5′-aagctGCCTTAGGCTTA(N)₁₅-3′ (SEQ ID NO:25) where theunderlined sequence is a Bsu36I site, the upper case nucleotides encodethe last four amino acids of the mature HA protein (ALGL), and (N)₁₅ isidentical to the first 15 nucleotides encoding the Therapetic protein ofinterest. Similarly, the reverse primer used to amplify the DNA encodinga Therapeutic protein might have the sequence,5′-GCGCGCGTTTAAACGGCCGGCCGGCGCGCCTTATTA(N)₁₅-3′

[0783] (SEQ ID NO:26) where the italicized nucleotides is a PmeI site,the double underlined nucleotides are a FseI site, the singly underlinedtext is a Pmel site, the boxed nucleotides are the reverse complement oftwo tandem stop codons, and (N)₁₅ is identical to the reverse complementof the last 15 nucleotides encoding the Therapeutic protein of interest.Once the PCR product is amplified it may be cut with Bsu361 and one of(AscI, FseI, or PmeI) and ligated into pScNHSA.

[0784] Making Vectors Comprising Albumin Fusion Proteins where theAlbumin Moiety is N-Terminal to the Therapeutic Moiety.

[0785] The DNA encoding the Therapeutic moiety may be PCR amplifiedusing primers that will add DNA encoding the last three amino acids ofthe Fusion leader sequence (and containing a SalI site) onto the 5′ endof the DNA encoding a Therapeutic protein and the first few amino acidsof the HA (and containing a ClaI site. For instance, the forward primerused to amplify the DNA encoding a Therapeutic protein might have thesequence, 5′-aggagcgtcGACAAAAGA(N)₅-3′ (SEQ ID NO:27) where theunderlined sequence is a Sal I site, the upper case nucleotides encodethe last three amino acids of the Fusion leader sequence (DKR), and(N)₁₅ is identical to the first 15 nucleotides encoding the Therapeticprotein of interest. Similarly, the reverse primer used to amplify theDNA encoding a Therapeutic protein might have the sequence,5′-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N)₁₅-3′ (SEQ ID NO:28) wherethe italicized nucleotides are a ClaI site, the underlined nucleotidesare the reverse complement of the DNA encoding the first 9 amino acidsof HA (DAHKSEVAH), and (N)₁₅ is identical to the reverse complement ofthe last 15 nucleotides encoding the Therapeutic protein of interest.Once the PCR product is amplified it may be cut with SalI and ClaI andligated into pScCHSA digested with XhoI and Cla I.

[0786] Expression of an Albumin Fusion Protein in Yeast.

[0787] The Not I fragment containing the DNA encoding either anN-terminal or C-terminal albumin fusion protein generated from pScCHSAor pScNHSA may then be cloned in to the NotI site of pSAC35.

[0788] Expression of an Albumin Fusion Protein from Mammalian Cell Lines

[0789] The HSA gene has also been cloned into a the pC4 vector which ismore suitable for mammalian culture systems creating plasmid pC4:HSA.More specifically, pC4HSA was generated by PCR amplifying the mature HSAgene with a 5′ primer (SEQ ID NO:30) that anneals to the 5′ end of DNAencoding the mature form of the HSA protein (e.g, DNA in plasmidpScCHSA),incorporates BamHI (Shown in italics below) and HindIII (shownsingly underlined below) cloning sites, attaches a kozak sequence (showndouble underlined below) and DNA encoding the natural HSA signal peptide(MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:29) (shown in bold below), and a 3′primer (SEQ ID NO:31) that anneals to the 3′ end of DNA encoding themature form of the HSA protein and incorporates an Asp718 restrictionsite (shown in bold below). The DNA encoding the natural human serumalbumin leader sequence in SEQ ID NO:30 also contains a modificationthat introduces a XhoI site that is boxed below. 5′-TCAGGGATCC AAGCTTCCGCCACCATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAG (SEQ ID NO:30)CTCGGCTTA{overscore (|CTCGAG|)}GGGTGTGTTTCGTCGAGATGCACACAAGAGTGAG-3′5″-GCAGCGGTACCGAATTCGGCGCGCCTTATAAGCCTAAGGCAGC-3′ (SEQ ID NO:31)

[0790] This PCR product (1.85 kb) is then purified and digested withBamHI and Asp718 and cloned into the same sites in pC4 (ATCC AccessionNo. 209646) to produce pC4:HSA

[0791] Making Vectors Comprising Albumin Fusion Proteins where theAlbumin Moiety is C-Terminal to the Therapeutic Moiety using the pC4:HSAVector

[0792] Using pC4:HSA, albumin fusion proteins in which the Therapeuticprotein moiety is N terminal to the albumin sequence, one can clone DNAencoding a Therapeutic protein that has its own signal sequence betweenthe Bam HI (or HindIII) and ClaI sites. When cloning into either theBamHI or Hind III site remember to include Kozak sequence (CCGCCACCATG)prior to translational start codon of DNA encoding the TherapeuticProtein to be subcloned. If the Therapeutic does not have a signalsequence, the DNA encoding that Therapeutic protein may be cloned inbetween the XhoI and ClaI sites. When using the XhoI site, the following5′ (SEQ ID NO:32) and 3′ (SEQ IDNO:33) PCR primers may be used: (SEQ IDNO:32) 5′-CCGCCGCTCGAGGGGTGTGTTTCGTCGA(N)₁₈-3′ (SEQ ID NO:33)5′-AGTCCCATCGATGAGCAACCTCACTTGTTGTGTGCATC(N)₁₈-3′

[0793] In SEQ ID NO:32, the underlined sequence is an XhoI site; and theXhoI site and the DNA following the XhoI site encode for the last sevenamino acids of the leader sequence of natural human serum albumin. InSEQ ID NO:33, the underlined sequence is a ClaI site; and the ClaI siteand the DNA following it encode are the reverse complement of the DNAencoding the first 10 amino acids of themature HSA protein (SEQ IDNO:18). In SEQ ID NO:32 “(N)₁₈” is DNA identical to the first 18nucleotides encoding the Therapeutic protein of interest.). In SEQ IDNO:33 “(N)₁₈” is the reverse complement of DNA encoding the last 18nucleotides encoding the Therapeutic protein of interest. Using thesetwo primers, one may PCR amplify the Therapeutic protein of interest,purify the PCR product, digest it with XhoI and ClaI restriction enzymesand then and clone it into the with XhoI and ClaI sites in the pC4:HSAvector.

[0794] Making Vectors Comprising Albumin Fusion Proteins where theAlbumin Moiety is N-Terminal to the Therapeutic Moiety using the pC4:HSAVector

[0795] Using pC4:HSA, albumin fusion proteins in which the Therapeuticprotein moiety is N terminal to the albumin sequence, one can clone DNAencoding a Therapeutic protein between the Bsu36I and AscI restrictionsites. When cloning into the Bsu36I and AscI, the same primer designused to clone in the yeast vector system (SEQ ID NO:25 and 26) may beemployed.

[0796] The pC4 vector is especially suitable for expression of albuminfusion proteins from CHO cells. For expression, in other mammalian celltypes, e.g., NSO cells, it may be useful to subclone the HindIII-EcoRIfragment containing the DNA encoding an albumin fusion protein (from apC4 vector in which the DNA encoding the Therapeutic protein has alreadybeen cloned in frame with the DNA encoding (the mature form of) humanserum albumin) into another expression vector (such as any of themammalian expression vectors described herein).

Example 3 Preparation of HA-Cytokine or HA-Growth Factor Fusion Proteins(such as EPO, GMCSF, GCSF)

[0797] The cDNA for the cytokine or growth factor of interest, such asEPO, can be isolated by a variety of means including from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for all of these proteins are known and available, forinstance, in U.S. Pat. Nos. 4,703,008, 4,810,643 and 5,908.763. The cDNAcan be tailored at the 5′ and 3′ ends to generate restriction sites,such that oligonucleotide linkers can be used, for cloning of the cDNAinto a vector containing the cDNA for HA. This can be at the N orC-terminus with or without the use of a spacer sequence. EPO (or othercytokine) cDNA is cloned into a vector such as pPPC0005 (FIG. 4),pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassetteis then excised and inserted into the plasmid pSAC35 to allow theexpression of the albumin fusion protein in yeast. The albumin fusionprotein secreted from the yeast can then be collected and purified fromthe media and tested for its biological activity. For expression inmammalian cell lines, a similar procedure is adopted except that theexpression cassette used employs a mammalian promoter, leader sequenceand terminator (See Example 2). This expression cassette is then excisedand inserted into a plasmid suitable for the transfection of mammaliancell lines.

Example 4 Preparation of HA-IFN Fusion Proteins (such as IFNα)

[0798] The cDNA for the interferon of interest such as IFNα can beisolated by a variety of means including but not exclusively, from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for interferons, such as IFNα are known and available, forinstance, in U.S. Pat. Nos. 5,326,859 and 4,588,585, in EP 32 134, aswell as in public databases such as GenBank. The cDNA can be tailored atthe 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used to clone the cDNA into a vectorcontaining the cDNA for HA. This can be at the N or C-terminus of the HAsequence, with or without the use of a spacer sequence. The IFNα (orother interferon) cDNA is cloned into a vector such as pPPC0005 (FIG.4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expressioncassette is then excised and inserted into the plasmid pSAC35 to allowthe expression of the albumin fusion protein in yeast (see FIG. 8). Thealbumin fusion protein secreted from the yeast can then be collected andpurified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines.

[0799] Maximum Protein Recovery from Vials

[0800] The albumin fusion proteins of the invention have a high degreeof stability even when they are packaged at low concentrations. Inaddition, in spite of the low protein concentration, good fusion-proteinrecovery is observed even when the aqueous solution includes no otherprotein added to minimize binding to the vial walls. FIG. 5 compares therecovery of vial-stored HA-IFN solutions with a stock solution. 6 or 30μg/ml HA-IFN solutions were placed in vials and stored at 4° C. After 48or 72 hrs a volume originally equivalent to 10 ng of sample was removedand measured in an IFN sandwich ELISA. The estimated values werecompared to that of a high concentration stock solution. As shown, thereis essentially no loss of the sample in these vials, indicating thataddition of exogenous material such as albumin is not necessary toprevent sample loss to the wall of the vials

[0801] In vivo Stability and Bioavailability of HA-α-IFN Fusions

[0802] To determine the in vivo stability and bloavailability of aHA-α-IFN fusion molecule, the purified fusion molecule (from yeast) wasadministered to monkeys at the dosages and time points described inFIGS. 6 and 7. Pharmaceutical compositions formulated from HA-α-IFNfusions may account for the extended serum half-life and bioavailabilityexemplified in FIGS. 6 and 7. Accordingly, pharmaceutical compositionsmay be formulated to contain lower dosages of alpha-interferon activitycompared to the native alpha-interferon molecule.

[0803] Pharmaceutical compositions containing HA-α-IFN fusions may beused to treat or prevent disease in patients with any disease or diseasestate that can be modulated by the administration of α-IFN. Suchdiseases include, but are not limited to, hairy cell leukemia, Kaposi'ssarcoma, genital and anal warts, chronic hepatitis B, chronic non-A,non-B hepatitis, in particular hepatitis C, hepatitis D, chronicmyelogenous leukemia, renal cell carcinoma, bladder carcinoma, ovarianand cervical carcinoma, skin cancers, recurrent respiratorpapillomatosis, non-Hodgkin's and cutaneous T-cell lymphomas, melanoma,multiple myeloma, AIDS, multiple sclerosis, gliobastoma, etc. (seeInterferon Alpha, In: AHFS Drug Information, 1997.

[0804] Accordingly, the invention includes pharmaceutical compositionscontaining a HA-α-IFN fusion protein, polypeptide or peptide formulatedwith the proper dosage for human administration. The invention alsoincludes methods of treating patients in need of such treatmentcomprising at least the step of administering a pharmaceuticalcomposition containing at least one HA-(—IFN fusion protein, polypeptideor peptide.

[0805] Bifunctional HA-_-IFN Fusions

[0806] The HA-α-IFN expression vector of FIG. 8 is modified to includean insertion for the expression of bifunctional HA-α-IFN fusionproteins. For instance, the cDNA for a second protein of interest may beinserted in frame downstream of the “rHA-IFN” sequence after the doublestop codon has been removed or shifted downstream of the codingsequence. In one version of a bifunctional HA-α-IFN fusion protein, anantibody or fragment against B-lymphocyte stimulator protein (GenBankAcc 4455139) or polypeptide may be fused to one end of the HA componentof the fusion molecule. This bifunctional protein is useful formodulating any immune response generated by the α-IFN component of thefusion.

Example 5 Preparation of HA-Hormone Fusion Protein (such as Insulin, LH,FSH)

[0807] The cDNA for the hormone of interest such as insulin can beisolated by a variety of means including but not exclusively, from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for all of these proteins are known and available, forinstance, in public databases such as GenBank. The cDNA can be tailoredat the 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used, for cloning of the cDNA into avector containing the cDNA for HA. This can be at the N or C-terminuswith or without the use of a spacer sequence. The hormone cDNA is clonedinto a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSAfrom which the complete expression cassette is then excised and insertedinto the plasmid pSAC35 to allow the expression of the albumin fusionprotein in yeast. The albumin fusion protein secreted from the yeast canthen be collected and purified from the media and tested for itsbiological activity. For expression in mammalian cell lines a similarprocedure is adopted except that the expression cassette used employs amammalian promoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 6 Preparation of HA-Soluble Receptor or HA-Binding ProteinFusion Protein such as HA-TNF Receptor

[0808] The cDNA for the soluble receptor or binding protein of interestsuch as TNF receptor can be isolated by a variety of means including butnot exclusively, from cDNA libraries, by RT-PCR and by PCR using aseries of overlapping synthetic oligonucleotide primers, all usingstandard methods. The nucleotide sequences for all of these proteins areknown and available, for instance, in GenBank. The cDNA can be tailoredat the 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used, for cloning of the cDNA into avector containing the cDNA for HA. This can be at the N or C-terminuswith or without the use of a spacer sequence. The receptor cDNA iscloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, orpC4:HSA from which the complete expression cassette is then excised andinserted into the plasmid pSAC35 to allow the expression of the albuminfusion protein in yeast. The albumin fusion protein secreted from theyeast can then be collected and purified from the media and tested forits biological activity. For expression in mammalian cell lines asimilar procedure is adopted except that the expression cassette usedemploys a mammalian promoter, leader sequence and terminator (SeeExample 2). This expression cassette is then excised and inserted into aplasmid suitable for the transfection of mammalian cell lines.

Example 7 Preparation of HA-Growth Factors such as HA-IGF-1 FusionProtein

[0809] The cDNA for the growth factor of interest such as IGF-1 can beisolated by a variety of means including but not exclusively, from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods (see GenBank Acc.No.NP_(—)000609). The cDNA can be tailored at the 5′ and 3′ ends togenerate restriction sites, such that oligonucleotide linkers can beused, for cloning of the cDNA into a vector containing the cDNA for HA.This can be at the N or C-terminus with or without the use of a spacersequence. The growth factor cDNA is cloned into a vector such aspPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the completeexpression cassette is then excised and inserted into the plasmid pSAC35to allow the expression of the albumin fusion protein in yeast. Thealbumin fusion protein secreted from the yeast can then be collected andpurified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines.

Example 8 Preparation of HA-Single Chain Antibody Fusion Proteins

[0810] Single chain antibodies are produced by several methods includingbut not limited to: selection from phage libraries, cloning of thevariable region of a specific antibody by cloning the cDNA of theantibody and using the flanking constant regions as the primer to clonethe variable region, or by synthesizing an oligonucleotide correspondingto the variable region of any specific antibody. The cDNA can betailored at the 5′ and 3′ ends to generate restriction sites, such thatoligonucleotide linkers can be used, for cloning of the cDNA into avector containing the cDNA for HA. This can be at the N or C-terminuswith or without the use of a spacer sequence. The cell cDNA is clonedinto a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSAfrom which the complete expression cassette is then excised and insertedinto the plasmid pSAC35 to allow the expression of the albumin fusionprotein in yeast.

[0811] In fusion molecules of the invention, the V_(H) and V_(L) can belinked by one of the following means or a combination thereof: a peptidelinker between the C-terminus of the V_(H) and the N-terminus of theV_(L); a Kex2p protease cleavage site between the V_(H) and V_(L) suchthat the two are cleaved apart upon secretion and then self associate;and cystine residues positioned such that the V_(H) and V_(L) can form adisulphide bond between them to link them together (see FIG. 14). Analternative option would be to place the V_(H) at the N-terminus of HAor an HA domain fragment and the V_(L) at the C-terminus of the HA or HAdomain fragment.

[0812] The albumin fusion protein secreted from the yeast can then becollected and purified from the media and tested for its activity. Forexpression in mammalian cell lines a similar procedure is adopted exceptthat the expression cassette used employs a mammalian promoter, leadersequence and terminator (See Example 2). This expression cassette isthen excised and inserted into a plasmid suitable for the transfectionof mammalian cell lines. The antibody produced in this manner can bepurified from media and tested for its binding to its antigen usingstandard immunochemical methods.

Example 9 Preparation of HA-Cell Adhesion Molecule Fusion Proteins

[0813] The cDNA for the cell adhesion molecule of interest can beisolated by a variety of means including but not exclusively, from cDNAlibraries, by RT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. The nucleotidesequences for the known cell adhesion molecules are known and available,for instance, in GenBank. The cDNA can be tailored at the 5′ and 3′ endsto generate restriction sites, such that oligonucleotide linkers can beused, for cloning of the cDNA into a vector containing the cDNA for HA.This can be at the N or C-terminus with or without the use of a spacersequence. The cell adhesion molecule cDNA is cloned into a vector suchas pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which thecomplete expression cassette is then excised and inserted into theplasmid pSAC35 to allow the expression of the albumin fusion protein inyeast. The albumin fusion protein secreted from the yeast can then becollected and purified from the media and tested for its biologicalactivity. For expression in mammalian cell lines a similar procedure isadopted except that the expression cassette used employs a mammalianpromoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 10 Preparation of Inhibitory Factors and Peptides as HA FusionProteins (such as HA-Antiviral, HA-Antibiotic, HA-Enzyme Inhibitor andHA-Anti-Allergic Proteins)

[0814] The cDNA for the peptide of interest such as an antibioticpeptide can be isolated by a variety of means including but notexclusively, from cDNA libraries, by RT-PCR and by PCR using a series ofoverlapping synthetic oligonucleotide primers, all using standardmethods. The cDNA can be tailored at the 5′ and 3′ ends to generaterestriction sites, such that oligonucleotide linkers can be used, forcloning of the cDNA into a vector containing the cDNA for HA. This canbe at the N or C-terminus with or without the use of a spacer sequence.The peptide cDNA is cloned into a vector such as pPPC0005 (FIG. 4),pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassetteis then excised and inserted into the plasmid pSAC35 to allow theexpression of the albumin fusion protein in yeast. The albumin fusionprotein secreted from the yeast can then be collected and purified fromthe media and tested for its biological activity. For expression inmammalian cell lines a similar procedure is adopted except that theexpression cassette used employs a mammalian promoter, leader sequenceand terminator (See Example 2). This expression cassette is then excisedand inserted into a plasmid suitable for the transfection of mammaliancell lines.

Example 11 Preparation of Targeted HA Fusion Proteins

[0815] The cDNA for the protein of interest can be isolated from cDNAlibrary or can be made synthetically using several overlappingoligonucleotides using standard molecular biology methods. Theappropriate nucleotides can be engineered in the cDNA to form convenientrestriction sites and also allow the attachment of the protein cDNA toalbumin cDNA similar to the method described for hGH. Also a targetingprotein or peptide cDNA such as single chain antibody or peptides, suchas nuclear localization signals, that can direct proteins inside thecells can be fused to the other end of albumin. The protein of interestand the targeting peptide is cloned into a vector such as pPPC0005 (FIG.4), pScCHSA, pScNHSA, or pC4:HSA which allows the fusion with albumincDNA. In this manner both N- and C-terminal end of albumin are fused toother proteins. The fused cDNA is then excised from pPPC0005 and isinserted into a plasmid such as pSAC35 to allow the expression of thealbumin fusion protein in yeast. All the above procedures can beperformed using standard methods in molecular biology. The albuminfusion protein secreted from yeast can be collected and purified fromthe media and tested for its biological activity and its targetingactivity using appropriate biochemical and biological tests.

Example 12 Preparation of HA-Enzymes Fusions

[0816] The cDNA for the enzyme of interest can be isolated by a varietyof means including but not exclusively, from cDNA libraries, by RT-PCRand by PCR using a series of overlapping synthetic oligonucleotideprimers, all using standard methods. The cDNA can be tailored at the 5′and 3′ ends to generate restriction sites, such that oligonucleotidelinkers can be used, for cloning of the cDNA into a vector containingthe cDNA for HA. This can be at the N or C-terminus with or without theuse of a spacer sequence. The enzyme cDNA is cloned into a vector suchas pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which thecomplete expression cassette is then excised and inserted into theplasmid pSAC35 to allow the expression of the albumin fusion protein inyeast. The albumin fusion protein secreted from the yeast can then becollected and purified from the media and tested for its biologicalactivity. For expression in mammalian cell lines a similar procedure isadopted except that the expression cassette used employs a mammalianpromoter, leader sequence and terminator (See Example 2). Thisexpression cassette is then excised and inserted into a plasmid suitablefor the transfection of mammalian cell lines.

Example 13 Bacterial Expression of an Albumin Fusion Protein

[0817] A polynucleotide encoding an albumin fusion protein of thepresent invention comprising a bacterial signal sequence is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′ ends ofthe DNA sequence, to synthesize insertion fragments. The primers used toamplify the polynucleotide encoding insert should preferably containrestriction sites, such as BamHI and XbaI, at the 5′ end of the primersin order to clone the amplified product into the expression vector. Forexample, BamHI and XbaI correspond to the restriction enzyme sites onthe bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth,Calif.). This plasmid vector encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatablepromoter/operator (P/O), a ribosome binding site (RBS), a 6-histidinetag (6-His), and restriction enzyme cloning sites.

[0818] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[0819] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[0820] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl or preferablyin 8 M urea and concentrations greater than 0.14 M 2-mercaptoethanol bystirring for 3-4 hours at 4° C. (see, e.g., Burton et al., Eur. J.Biochem. 179:379-387 (1989)). The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[0821] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[0822] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. Exemplary conditions are asfollows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20%glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

[0823] In addition to the above expression vector, the present inventionfurther includes an expression vector, called pHE4a (ATCC AccessionNumber 209645, deposited on Feb. 25, 1998) which contains phage operatorand promoter elements operatively linked to a polynucleotide encoding analbumin fusion protein of the present invention, called pHE4a. (ATCCAccession Number 209645, deposited on Feb. 25, 1998.) This vectorcontains: 1) a neomycinphosphotransferase gene as a selection marker, 2)an E. coli origin of replication, 3) a T5 phage promoter sequence, 4)two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) thelactose operon repressor gene (lacIq). The origin of replication (oriC)is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter andoperator sequences are made synthetically.

[0824] DNA can be inserted into the pHE4a by restricting the vector withNdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product ona gel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to PCRprotocols described herein or otherwise known in the art, using PCRprimers having restriction sites for NdeI (5′ primer) and XbaI, BamHI,XhoI, or Asp718 (3′ primer). The PCR insert is gel purified andrestricted with compatible enzymes. The insert and vector are ligatedaccording to standard protocols.

[0825] The engineered vector may be substituted in the above protocol toexpress protein in a bacterial system.

Example 14 Expression of an Albumin Fusion Protein in Mammalian Cells

[0826] The albumin fusion proteins of the present invention can beexpressed in a mammalian cell. A typical mammalian expression vectorcontains a promoter element, which mediates the initiation oftranscription of mRNA, a protein coding sequence, and signals requiredfor the termination of transcription and polyadenylation of thetranscript. Additional elements include enhancers, Kozak sequences andintervening sequences flanked by donor and acceptor sites for RNAsplicing. Highly efficient transcription is achieved with the early andlate promoters from SV40, the long terminal repeats (LTRs) fromRetroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of thecytomegalovirus (CMV). However, cellular elements can also be used(e.g., the human actin promoter).

[0827] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as, pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, but are not limited to,human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamsterovary (CHO) cells.

[0828] Alternatively, the albumin fusion protein can be expressed instable cell lines containing the polynucleotide encoding the albuminfusion protein integrated into a chromosome. The co-transfection with aselectable marker such as DHFR, gpt, neomycin, or hygromycin allows theidentification and isolation of the transfected cells.

[0829] The transfected polynucleotide encoding the fusion protein canalso be amplified to express large amounts of the encoded fusionprotein. The DHFR (dihydrofolate reductase) marker is useful indeveloping cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt et al., J.Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et Biophys.Acta, 1097:107-143 (1990); Page et al., Biotechnology 9:64-68 (1991)).Another useful selection marker is the enzyme glutamine synthase (GS)(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,Bio/Technology 10:169-175 (1992). Using these markers, the mammaliancells are grown in selective medium and the cells with the highestresistance are selected. These cell lines contain the amplified gene(s)integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cellsare often used for the production of proteins.

[0830] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146),the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

[0831] Specifically, the plasmid pC6, for example, is digested withappropriate restriction enzymes and then dephosphorylated using calfintestinal phosphates by procedures known in the art. The vector is thenisolated from a 1% agarose gel.

[0832] A polynucleotide encoding an albumin fusion protein of thepresent invention is generated using techniques known in the art andthis polynucleotide is amplified using PCR technology known in the art.If a naturally occurring signal sequence is used to produce the fusionprotein of the present invention, the vector does not need a secondsignal peptide. Alternatively, if a naturally occurring signal sequenceis not used, the vector can be modified to include a heterologous signalsequence. (See, e.g., International Publication No. WO 96/34891.)

[0833] The amplified fragment encoding the fusion protein of theinvention is isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment then is digested with appropriate restriction enzymes and againpurified on a 1% agarose gel.

[0834] The amplified fragment encoding the albumin fusion protein of theinvention is then digested with the same restriction enzyme and purifiedon a 1% agarose gel. The isolated fragment and the dephosphorylatedvector are then ligated with T4 DNA ligase. E. coli HB11 or XL-1 Bluecells are then transformed and bacteria are identified that contain thefragment inserted into plasmid pC6 using, for instance, restrictionenzyme analysis.

[0835] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 μg of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ngiml of methotrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 μM, 2 μM, 5 ₁ μM, 10 mM, 20mM). The same procedure is repeated until clones are obtained which growat a concentration of 100-200 μM. Expression of the desired fusionprotein is analyzed, for instance, by SDS-PAGE and Western blot or byreversed phase HPLC analysis.

Example 15 Multifusion Fusions

[0836] The albumin fusion proteins (e.g,. containing a Therapeuticprotein (or fragment or variant thereof) fused to albumin (or a fragmentor variant thereof)) may additionally be fused to other proteins togenerate “multifusion proteins”. These multifusion proteins can be usedfor a variety of applications. For example, fusion of the albumin fusionproteins of the invention to His-tag, HA-tag, protein A, IgG domains,and maltose binding protein facilitates purification. (See e.g,. EP A394,827; Traunecker et al., Nature 331:84-86 (1988)). Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof an albumin fusion protein. Furthermore, the fusion of additionalprotein sequences to the albumin fusion proteins of the invention mayfurther increase the solubility and/or stability of the fusion protein.The fusion proteins described above can be made using or routinelymodifting techniques known in the art and/or by modifying the followingprotocol, which outlines the fusion of a polypeptide to an IgG molecule.

[0837] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian or yeast expression vector.

[0838] For example, if pC4 (ATCC Accession No. 209646) is used, thehuman Fc portion can be ligated into the BamHil cloning site. Note thatthe 3′ BamHI site should be destroyed. Next, the vector containing thehuman Fc portion is re-restricted with BamHI, linearizing the vector,and a polynucleotide encoding an albumin fusion protein of the presentinvention (generateed and isolated using techniques known in the art),is ligated into this BamHI site. Note that the polynucleotide encodingthe fusion protein of the invention is cloned without a stop codon,otherwise a Fc containing fusion protein will not be produced.

[0839] If the naturally occurring signal sequence is used to produce thealbumin fusion protein of the present invention, pC4 does not need asecond signal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., International Publication No.WO 96/34891.)

[0840] Human IgG Fc Region: (SEQ ID NO: 36)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCFGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 16 Production of an Antibody from an Albumin Fusion Protein

[0841] a) Hybridoma Technology

[0842] Antibodies that bind the albumin fusion proteins of the presentinvention and portions of the albumin fusion proteins of the presentinvention (e.g., the Therapeutic protein portion or albumin portion ofthe fusion protein) can be prepared by a variety of methods. (See,Current Protocols, Chapter 2.) As one example of such methods, apreparation of an albumin fusion protein of the invention or a portionof an albumin fusion protein of the invention is prepared and purifiedto render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[0843] Monoclonal antibodies specific for an albumin fusion protein ofthe invention, or a portion of an albumin fusion protein of theinvention, are prepared using hybridoma technology (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, an animal (preferably a mouse) is immunized with analbumin fusion protein of the invention, or a portion of an albuminfusion protein of the invention. The splenocytes of such mice areextracted and fused with a suitable myeloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; however, it is preferable to employ the parent myeloma cellline (SP20), available from the ATCC. After fusion, the resultinghybridoma cells are selectively maintained in HAT medium, and thencloned by limiting dilution as described by Wands et al.(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtainedthrough such a selection are then assayed to identify clones whichsecrete antibodies capable of binding an albumin fusion protein of theinvention, or a portion of an albumin fusion protein of the invention.

[0844] Alternatively, additional antibodies capable of binding to analbumin fusion protein of the invention, or a portion of an albuminfusion protein of the invention can be produced in a two-step procedureusing anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thean albumin fusion protein of the invention (or portion of an albuminfusion protein of the invention)-specific antibody can be blocked by thefusion protein of the invention, or a portion of an albumin fusionprotein of the invention. Such antibodies comprise anti-idiotypicantibodies to the fusion protein of the invention (or portion of analbumin fusion protein of the invention)-specific antibody and are usedto immunize an animal to induce formation of further fusion protein ofthe invention (or portion of an albumin fusion protein of theinvention)-specific antibodies.

[0845] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., International Publication No. WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).

[0846] b) Isolation of Antibody Fragments Directed Against an AlbuminFusion Protein of the Invention, or a Portion of an Albumin FusionProtein of the Invention from a Library of scFvs

[0847] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against an albumin fusion protein of the invention, or aportion of an albumin fusion protein of the invention, to which thedonor may or may not have been exposed (see e.g., U.S. Pat. No.5,885,793 incorporated herein by reference in its entirety).

[0848] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in International Publication No. WO92/01047. To rescue phage displaying antibody fragments, approximately10⁹ E. coli harboring the phagemid are used to inoculate 50 ml of 2×TYcontaining 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) andgrown to an O.D. of 0.8 with shaking. Five ml of this culture is used toinoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13delta gene III, see International Publication No. WO 92/01047) are addedand the culture incubated at 37° C. for 45 minutes without shaking andthen at 37° C. for 45 minutes with shaking. The culture is centrifugedat 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phage are prepared as described in International PublicationNo. WO 92/01047.

[0849] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 10¹³transducing units/ml (ampicillin-resistant clones).

[0850] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 pg/ml of an albumin fusionprotein of the invention, or a portion of an albumin fusion protein ofthe invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C. and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 nl of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG I by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0851] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of analbumin fusion protein of the invention, or a portion of an albuminfusion protein of the invention, in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., International Publication No. WO 92/01047) and then by sequencing.These ELISA positive clones may also be further characterized bytechniques known in the art, such as, for example, epitope mapping,binding affinity, receptor signal transduction, ability to block orcompetitively inhibit antibody/antigen binding, and competitiveagonistic or antagonistic activity.

Example 17 Method of Treatment Using Gene Therapy-Ex Vivo

[0852] One method of gene therapy transplants fibroblasts, which arecapable of expressing an albumin fusion protein of the presentinvention, onto a patient. Generally, fibroblasts are obtained from asubject by skin biopsy. The resulting tissue is placed in tissue-culturemedium and separated into small pieces. Small chunks of the tissue areplaced on a wet surface of a tissue culture flask, approximately tenpieces are placed in each flask. The flask is turned upside down, closedtight and left at room temperature over night. After 24 hours at roomtemperature, the flask is inverted and the chunks of tissue remain fixedto the bottom of the flask and fresh media (e.g., Ham's F12 media, with10% FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37 degree C. for approximately one week.

[0853] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks. pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0854] Polynucleotides encoding an albumin fusion protein of theinvention can be generated using techniques known in the art amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequences andoptionally having appropriate restriction sites and initiation/stopcodons, if necessary. Preferably, the 5′ primer contains an EcoRI siteand the 3′ primer includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[0855] The amphotropic pA317 or GP+aml2 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0856] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether the albumin fusion protein is produced.

[0857] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 18 Method of Treatment Using Gene Therapy—In Vivo

[0858] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences encoding an albumin fusionprotein of the invention into an animal. Polynucleotides encodingalbumin fusion proteins of the present invention may be operativelylinked to (i.e., associated with) a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[0859] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0860] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, polynucleotides encoding albumin fusion proteins ofthe present invention may also be delivered in liposome formulations(such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci.772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) whichcan be prepared by methods well known to those skilled in the art.

[0861] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapy techniques, onemajor advantage of introducing naked nucleic acid sequences into targetcells is the transitory nature of the polynucleotide synthesis in thecells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[0862] The polynucleotide construct can be delivered to the interstitialspace of tissues within an animal, including muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0863] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[0864] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[0865] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[0866] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for fusion proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of DNA inmuscle following injection may be determined by Southern blot analysisafter preparing total cellular DNA and HIRT supernatants from injectedand control mice. The results of the above experimentation in mice canbe used to extrapolate proper dosages and other treatment parameters inhumans and other animals using naked DNA.

Example 19 Transgenic Animals

[0867] The albumin fusion proteins of the invention can also beexpressed in transgenic animals. Animals of any species, including, butnot limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons,monkeys, and chimpanzees may be used to generate transgenic animals. Ina specific embodiment, techniques described herein or otherwise known inthe art, are used to express fusion proteins of the invention in humans,as part of a gene therapy protocol.

[0868] Any technique known in the art may be used to introduce thepolynucleotides encoding the albumin fusion proteins of the inventioninto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to, pronuclear microinjection(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carveret al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al.,Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No.4,873,191 (1989)); retrovirus mediated gene transfer into germ lines(Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152(1985)), blastocysts or embryos; gene targeting in embryonic stem cells(Thompson et al., Cell 56:313-321 (1989)); electroporation of cells orembryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction ofthe polynucleotides of the invention using a gene gun (see, e.g., Ulmeret al., Science 259:1745 (1993); introducing nucleic acid constructsinto embryonic pleuripotent stem cells and transferring the stem cellsback into the blastocyst; and sperm-mediated gene transfer (Lavitrano etal., Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[0869] Any technique known in the art may be used to produce transgenicclones containing polynucleotides encoding albumin fusion proteins ofthe invention, for example, nuclear transfer into enucleated oocytes ofnuclei from cultured embryonic, fetal, or adult cells induced toquiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al.,Nature 385:810-813 (1997)).

[0870] The present invention provides for transgenic animals that carrythe polynucleotides encoding the albumin fusion proteins of theinvention in all their cells, as well as animals which carry thesepolynucleotides in some, but not all their cells, i.e., mosaic animalsor chimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide encoding the fusion protein of theinvention be integrated into the chromosomal site of the endogenous genecorresponding to the Therapeutic protein portion or ablumin portion ofthe fusion protein of the invention, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0871] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thepolynucleotide encoding the fsuion protien of the invention has takenplace. The level of mRNA expression of the polynucleotide encoding thefusion protein of the invention in the tissues of the transgenic animalsmay also be assessed using techniques which include, but are not limitedto, Northern blot analysis of tissue samples obtained from the animal,in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR).Samples of fusion protein-expressing tissue may also be evaluatedimmunocytochemically or immunohistochemically using antibodies specificfor the fusion protein.

[0872] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene(i.e., polynucleotide encoding an albumin fusion protein of theinvention) on a distinct background that is appropriate for anexperimental model of interest.

[0873] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of fusion proteins of the invention and theTherapeutic protein and/or albumin component of the fusion protein ofthe invention, studying conditions and/or disorders associated withaberrant expression, and in screening for compounds effective inameliorating such conditions and/or disorders.

Example 20 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

[0874] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL-10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[0875] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[0876] In Vitro Assay—Albumin fusion proteins of the invention(including fusion proteins containing fragments or variants ofTherapeutic proteins and/or albumin or fragments or variants of albumin)can be assessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of an albumin fusion protein of theinvention on purified human tonsillar B cells, measured qualitativelyover the dose range from 0.1 to 10,000 ng/mL, is assessed in a standardB-lymphocyte co-stimulation assay in which purified tonsillar B cellsare cultured in the presence of either formalin-fixed Staphylococcusaureus Cowan I (SAC) or immobilized anti-human IgM antibody as thepriming agent. Second signals such as IL-2 and IL-15 synergize with SACand IgM crosslinking to elicit B cell proliferation as measured bytritiated-thymidine incorporation. Novel synergizing agents can bereadily identified using this assay. The assay involves isolating humantonsillar B cells by magnetic bead (MACS) depletion of CD3-positivecells. The resulting cell population is greater than 95% B cells asassessed by expression of CD45R(B220).

[0877] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 10⁵ B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100U/ml penicillin,10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[0878] In vivo Assay—BALB/c mice are injected (i.p.) twice per day withbuffer only, or 2 mg/Kg of an albumin fusion protein of the invention(including fusion proteins containing fragments or variants ofTherapeutic proteins and/or albumin or fragments or variants ofalbumin). Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with the albumin fusion protein of the invention identify theresults of the activity of the fusion protein on spleen cells, such asthe diffusion of periarterial lymphatic sheaths, and/or significantincreases in the nucleated cellularity of the red pulp regions, whichmay indicate the activation of the differentiation and proliferation ofB-cell populations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[0879] Flow cytometric analyses of the spleens from mice treated withthe albumin fusion protein is used to indicate whether the albuminfusion protein specifically increases the proportion of ThB+,CD45R(B220)dull B cells over that which is observed in control mice.

[0880] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andfusion protein treated mice.

[0881] The studies described in this example tested activity of fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinsand polynucleotides of the invention (e.g., gene therapy).

Example 21 T Cell Proliferation Assay

[0882] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of ³H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 μl/well of mAb toCD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnightat 4 degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), thenwashed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of an albumin fusionprotein of the invention (including fusion proteins containing fragmentsor variants of Therapeutic proteins and/or albumin or fragments orvariants of albumin) (total volume 200 ul). Relevant protein buffer andmedium alone are controls. After 48 hr, culture at 37 degrees C., platesare spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed andstored −20 degrees C. for measurement of IL-2 (or other cytokines) ifeffect on proliferation is observed. Wells are supplemented with 100 ulof medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degreesC. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidineused as a measure of proliferation. Anti-CD3 alone is the positivecontrol for proliferation. IL-2 (100 U/ml) is also used as a controlwhich enhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative control for the effectsof fusion proteins of the invention.

[0883] The studies described in this example tested activity of fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinsor polynucleotides of the invention (e.g., gene therapy).

Example 22 Effect of Fusion Proteins of the Invention on the Expressionof MHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

[0884] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α:,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγ R11, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

[0885] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of an albumin fusionprotein of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate HTC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

[0886] Effect on the production of cytokines. Cytokines generated bydendritic cells, in particular IL-12, are important in the initiation ofT-cell dependent immune responses. IL-12 strongly influences thedevelopment of Th1 helper T-cell immune response, and induces cytotoxicT and NK cell function. An ELISA is used to measure the IL-12 release asfollows. Dendritic cells (10⁶/ml) are treated with increasingconcentrations of an albumin fusion protein of the invention for 24hours. LPS (100 ng/ml) is added to the cell culture as positive control.Supernatants from the cell cultures are then collected and analyzed forIL-12 content using commercial ELISA kit (e.g., R & D Systems(Minneapolis, Minn.)). The standard protocols provided with the kits areused.

[0887] Effect on the expression of MHC Class II, costimulatory andadhesion molecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increased expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[0888] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of analbumin fusion protein of the invention or LPS (positive control),washed with PBS containing 1% BSA and 0.02 mM sodium azide, and thenincubated with 1:20 dilution of appropriate FITC— or PE-labeledmonoclonal antibodies for 30 minutes at 4 degrees C. After an additionalwash, the labeled cells are analyzed by flow cytometry on a FACScan(Becton Dickinson).

[0889] Monocyte activation and/or increased survival. Assays formolecules that activate (or alternatively, inactivate) monocytes and/orincrease monocyte survival (or alternatively, decrease monocytesurvival) are known in the art and may routinely be applied to determinewhether a molecule of the invention functions as an inhibitor oractivator of monocytes. Albumin fusion proteins of the invention can bescreened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

[0890] Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated processes(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the fusion protein to be tested. Cells aresuspended at a concentration of 2×10⁶/ml in PBS containing PI at a finalconcentration of 5 μg/ml, and then incubated at room temperature for 5minutes before FACScan analysis. PI uptake has been demonstrated tocorrelate with DNA fragmentation in this experimental paradigm.

[0891] Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of an albumin fusion protein of theinvention and under the same conditions, but in the absence of thefusion protein. For IL-12 production, the cells are primed overnightwith IFN (100 U/ml) in the presence of the fusion protein. LPS (10ng/ml) is then added. Conditioned media are collected after 24 h andkept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8is then performed using a commercially available ELISA kit (e.g., R & DSystems (Minneapolis, Minn.)) and applying the standard protocolsprovided with the kit.

[0892] Oxidative burst. Purified monocytes are plated in 96-w plate at2-1×10⁵ cell/well. Increasing concentrations of an albumin fusionprotein of the invention are added to the wells in a total volume of 0.2ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After3 days incubation, the plates are centrifuged and the medium is removedfrom the wells. To the macrophage monolayers, 0.2 ml per well of phenolred solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37° C. for2 hours and the reaction is stopped by adding 20 yl 1N NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H₂O₂produced by the macrophages, a standard curve of a H₂O₂ solution ofknown molarity is performed for each experiment.

[0893] The studies described in this example tested activity of fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinsor polynucleotides of the invention (e.g., gene therapy).

Example 23 Biological Effects of Fusion Proteins of the Invention

[0894] Astrocyte and Neuronal Assays

[0895] Albumin fusion proteins of the invention can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FGF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate an albumin fusion protein of theinvention's activity on these cells.

[0896] Moreover, previous reports describing the biological effects ofFGF-2 (basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of an albuminfusion protein of the invention to induce neurite outgrowth can becompared to the response achieved with FGF-2 using, for example, athymidine incorporation assay.

[0897] Fibroblast and Endothelial Cell Assays

[0898] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest fusion protein of the invention proteins for 3 days. Alamar Blue(Alamar Biosciences, Sacramento, Calif.) is added to each well to afinal concentration of 10%. The cells are incubated for 4 hr. Cellviability is measured by reading in a CytoFluor fluorescence reader. Forthe PGE₂ assays, the human lung fibroblasts are cultured at 5,000cells/well in a 96-well plate for one day. After a medium change to 0.1%BSA basal medium, the cells are incubated with FGF-2 or fusion proteinof the invention with or without IL-1a for 24 hours. The supernatantsare collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor,Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at5,000 cells/well in a 96-well plate for one day. After a medium changeto 0.1% BSA basal medium, the cells are incubated with FGF-2 or with orwithout an albumin fusion protein of the invention and/or IL-1α for 24hours. The supernatants are collected and assayed for IL-6 by ELISA kit(Endogen, Cambridge, Mass.).

[0899] Human lung fibroblasts are cultured with FGF-2 or an albuminfusion protein of the invention for 3 days in basal medium before theaddition of Alamar Blue to assess effects on growth of the fibroblasts.FGF-2 should show a stimulation at 10-2500 ng/ml which can be used tocompare stimulation with the fusion protein of the invention.

[0900] Cell Proliferation Based on [3H]Thymidine Incorporation

[0901] The following [3H]Thymidine incorporation assay can be used tomeasure the effect of a Therapeutic proteins, e.g., growth factorproteins, on the proliferation of cells such as fibroblast cells,epithelial cells or immature muscle cells.

[0902] Sub-confluent cultures are arrested in GI phase by an 18 hincubation in serum-free medium. Therapeutic proteins are then added for24 h and during the last 4 h, the cultures are labeled with[3H]thymidine, at a final concentration of 0.33 μM (25 Ci/mmol,Amersham, Arlington Heights, Ill.). The incorporated [3H]thymidine isprecipitated with ice-cold 10% trichloroacetic acid for 24 h.Subsequently, the cells are rinsed sequentially with ice-cold 10%trichloroacetic acid and then with ice-cold water. Following lysis in0.5 M NaOH, the lysates and PBS rinses (500 ml) are pooled, and theamount of radioactivity is measured.

[0903] Parkinson Models.

[0904] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPITP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[0905] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[0906] Based on the data with FGF-2, an albumin fusion protein of theinvention can be evaluated to determine whether it has an action similarto that of FGF-2 in enhancing dopaminergic neuronal survival in vitroand it can also be tested in vivo for protection of dopaminergic neuronsin the striatum from the damage associated with MPTP treatment. Thepotential effect of an albumin fusion protein of the invention is firstexamined in vitro in a dopaminergic neuronal cell culture paradigm. Thecultures are prepared by dissecting the midbrain floor plate fromgestation day 14 Wistar rat embryos. The tissue is dissociated withtrypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells are maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (NI). The cultures are fixed with paraformaldehyde after 8days in vitro and are processed for tyrosine hydroxylase, a specificmarker for dopaminergic neurons, immunohistochemical staining.Dissociated cell cultures are prepared from embryonic rats. The culturemedium is changed every third day and the factors are also added at thattime.

[0907] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a Therapeutic protein acts to prolong the survival ofdopaminergic neurons, it would suggest that the fusion protein may beinvolved in Parkinson's Disease.

[0908] The studies described in this example tested activity of albuminfusion proteins of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of fusionproteins and polynucleotides of the invention (e.g., gene therapy).

Example 24 The Effect of Albumin Fusion Proteins of the Invention on theGrowth of Vascular Endothelial Cells

[0909] On day 1, human umbilical vein endothelial cells (HUVEC) areseeded at 2-5×10o cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium is replaced with M199 containing 10% FBS, 8 units/mlheparin. An albumin fusion protein of the invention, and positivecontrols, such as VEGF and basic FGF (bFGF) are added, at varyingconcentrations. On days 4 and 6, the medium is replaced. On day 8, cellnumber is determined with a Coulter Counter.

[0910] An increase in the number of HUVEC cells indicates that thefusion protein may proliferate vascular endothelial cells, while adecrease in the number of HUVEC cells indicates that the fusion proteininhibits vascular endothelial cells.

[0911] The studies described in this example tested activity of analbumin fusion protein of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity of afusion protiem and polynucleotides of the invention.

Example 25 Rat Corneal Wound Healing Model

[0912] This animal model shows the effect of an albumin fusion proteinof the invention on neovascularization. The experimental protocolincludes:

[0913] Making a 1-1.5 mm long incision from the center of cornea intothe stromal layer.

[0914] Inserting a spatula below the lip of the incision facing theouter corner of the eye.

[0915] Making a pocket (its base is 1-1.5 mm form the edge of the eye).

[0916] Positioning a pellet, containing 50 ng-5 ug of an albumin fusionprotein of the invention, within the pocket.

[0917] Treatment with an an albumin fusion protein of the invention canalso be applied topically to the corneal wounds in a dosage range of 20mg-500 mg (daily treatment for five days).

[0918] The studies described in this example test the activity of analbumin fusion protein of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity offusion proteins and polynucleotides of the invention (e.g., genetherapy).

Example 26 Diabetic Mouse and Glucocorticoid-Impaired

[0919] Wound Healing Models

[0920] Diabetic db+/db+ Mouse Model.

[0921] To demonstrate that an albumin fusion protein of the inventionaccelerates the healing process, the genetically diabetic mouse model ofwound healing is used. The full thickness wound healing model in thedb+/db+ mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

[0922] The diabetic animals have many of the characteristic featuresobserved in Type II diabetes mellitus. Homozygous (db+/db+) mice areobese in comparison to their normal heterozygous (db+/+m) littermates.Mutant diabetic (db+/db+) mice have a single autosomal recessivemutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci.USA 77:283-293 (1982)). Animals show polyphagia, polydipsia andpolyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,increased or normal insulin levels, and suppressed cell-mediatedimmunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M.et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardialcomplications, and microvascular lesions, basement membrane thickeningand glomerular filtration abnormalities have been described in theseanimals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertsonet al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)).These homozygous diabetic mice develop hyperglycemia that is resistantto insulin analogous to human type II diabetes (Mandel et al., J.Immunol. 120:1375-1377 (1978)).

[0923] The characteristics observed in these animals suggests thathealing in this model may be similar to the healing observed in humandiabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

[0924] Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

[0925] Wounding protocol is performed according to previously reportedmethods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

[0926] Wounds are visually examined and photographed at a fixed distanceat the day of surgery and at two day intervals thereafter. Wound closureis determined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[0927] An albumin fusion protein of the invention is administered usingat a range different doses, from 4 mg to 500 mg per wound per day for 8days in vehicle. Vehicle control groups received 50 mL of vehiclesolution.

[0928] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

[0929] Three groups of 10 animals each (5 diabetic and 5 non-diabeticcontrols) are evaluated: 1) Vehicle placebo control, 2) untreated group,and 3) treated group.

[0930] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0931] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with an albumin fusion protein of the invention. Thisassessment included verification of the presence of cell accumulation,inflammatory cells, capillaries, fibroblasts, re-epithelialization andepidermal maturity (Greenhaigh, D. G. et al., Am. J. Pathol. 136.1235(1990)). A calibrated lens micrometer is used by a blinded observer.

[0932] Tissue sections are also stained immunohistochemically with apolyclonal rabbit anti-human keratin antibody using ABC Elite detectionsystem. Human skin is used as a positive tissue control while non-immuneIgG is used as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

[0933] Proliferating cell nuclear antigen/cyclin (PCNA) in skinspecimens is demonstrated by using anti-PCNA antibody (1:50) with an ABCElite detection system. Human colon cancer served as a positive tissuecontrol and human brain tissue is used as a negative tissue control.Each specimen included a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

[0934] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[0935] Steroid Impaired Rat Model

[0936] The inhibition of wound healing by steroids has been welldocumented in various in vitro and in vivo systems (Wahl,Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action:Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115:476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin.Invest. 61: 703-797 (1978)) and producing a transient reduction ofcirculating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797(1978); Wahl, “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

[0937] To demonstrate that an albumin fusion protein of the inventioncan accelerate the healing process, the effects of multiple topicalapplications of the fusion protein on full thickness excisional skinwounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

[0938] Young adult male Sprague Dawley rats weighing 250-300 g (CharlesRiver Laboratories) are used in this example. The animals are purchasedat 8 weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals,are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

[0939] The wounding protocol is followed according to that describedabove. On the day of wounding, animals are anesthetized with anintramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg).The dorsal region of the animal is shaved and the skin washed with 70%ethanol and iodine solutions. The surgical area is dried with sterilegauze prior to wounding. An 8 mm full-thickness wound is created using aKeyes tissue punch. The wounds are left open for the duration of theexperiment. Applications of the testing materials are given topicallyonce a day for 7 consecutive days commencing on the day of wounding andsubsequent to methylprednisolone administration. Prior to treatment,wounds are gently cleansed with sterile saline and gauze sponges.

[0940] Wounds are visually examined and photographed at a fixed distanceat the day of wounding and at the end of treatment. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[0941] The fusion protein of the invention is administered using at arange different doses, from 4 mg to 500 mg per wound per day for 8 daysin vehicle. Vehicle control groups received 50 mL of vehicle solution.

[0942] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

[0943] Three groups of 10 animals each (5 with methylprednisolone and 5without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicleplacebo control 3) treated groups.

[0944] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total area of the wound. Closureis then estimated by establishing the differences between the initialwound area (day 0) and that of post treatment (day 8). The wound area onday 1 is 64 mm², the corresponding size of the dermal punch.Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0945] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds allows assessment of whether the healingprocess and the morphologic appearance of the repaired skin is improvedby treatment with an albumin fusion protein of the invention. Acalibrated lens micrometer is used by a blinded observer to determinethe distance of the wound gap.

[0946] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[0947] The studies described in this example tested activity of analbumin fusion protein of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity offusion proteins and polynucleotides of the invention (e.g., genetherapy).

Example 27 Lymphedema Animal Model

[0948] The purpose of this experimental approach is to create anappropriate and consistent lymphedema model for testing the therapeuticeffects of an albumin fusion protein of the invention inlymphangiogenesis and re-establishment of the lymphatic circulatorysystem in the rat hind limb. Effectiveness is measured by swellingvolume of the affected limb, quantification of the amount of lymphaticvasculature, total blood plasma protein, and histopathology. Acutelymphedema is observed for 7-10 days. Perhaps more importantly, thechronic progress of the edema is followed for up to 3-4 weeks.

[0949] Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately 350 g are dosedwith Pentobarbital. Subsequently, the right legs are shaved from knee tohip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood isdrawn for serum total protein testing. Circumference and volumetricmeasurements are made prior to injecting dye into paws after marking 2measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). Theintradermal dorsum of both right and left paws are injected with 0.05 mlof 1% Evan's Blue. Circumference and volumetric measurements are thenmade following injection of dye into paws.

[0950] Using the knee joint as a landmark, a mid-leg inguinal incisionis made circumferentially allowing the femoral vessels to be located.Forceps and hemostats are used to dissect and separate the skin flaps.After locating the femoral vessels, the lymphatic vessel that runs alongside and underneath the vessel(s) is located. The main lymphatic vesselsin this area are then electrically coagulated or suture ligated.

[0951] Using a microscope, muscles in back of the leg (near thesemitendinosis and adductors) are bluntly dissected. The popliteal lymphnode is then located. The 2 proximal and 2 distal lymphatic vessels anddistal blood supply of the popliteal node are then ligated by suturing.The popliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

[0952] Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of 0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

[0953] To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effect ofplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

[0954] Circumference Measurements: Under brief gas anesthetic to preventlimb movement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople and those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

[0955] Volumetric Measurements: On the day of surgery, animals areanesthetized with Pentobarbital and are tested prior to surgery. Fordaily volumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), and both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

[0956] Blood-plasma protein measurements: Blood is drawn, spun, andserum separated prior to surgery and then at conclusion for totalprotein and Ca2+ comparison.

[0957] Limb Weight Comparison: After drawing blood, the animal isprepared for tissue collection. The limbs are amputated using aquillitine, then both experimental and control legs are cut at theligature and weighed. A second weighing is done as the tibio-cacanealjoint is disarticulated and the foot is weighed.

[0958] Histological Preparations: The transverse muscle located behindthe knee (popliteal) area is dissected and arranged in a metal mold,filled with freezeGel, dipped into cold methylbutane, placed intolabeled sample bags at −80EC until sectioning. Upon sectioning, themuscle is observed under fluorescent microscopy for lymphatics.

[0959] The studies described in this example tested activity of fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinand polynucleotides of the invention (e.g., gene therapy).

Example 28 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby an Albumin Fusion Protein of the Invention

[0960] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[0961] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[0962] The potential of an albumin fusion protein of the invention tomediate a suppression of TNF-a induced CAM expression can be examined. Amodified ELISA assay which uses ECs as a solid phase absorbent isemployed to measure the amount of CAM expression on TNF-a treated ECswhen co-stimulated with a member of the FGF family of proteins.

[0963] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% CO₂. HUVECs are seeded in 96-well plates atconcentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPMI-1640 supplemented with 100 U/mlpenicillin and 100 mg/ml streptomycin, and treated with a given cytokineand/or growth factor(s) for 24 h at 37 degree C. Following incubation,the cells are then evaluated for CAM expression.

[0964] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and 100 μl of0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well.Plates are held at 4° C. for 30 min.

[0965] Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

[0966] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase(1:5,000 dilution) to each well and incubated at 37° C. for 30 min.Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in gJycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5).5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

[0967] The studies described in this example tested activity of fusionproteins of the invention. However, one skilled in the art could easilymodify the exemplified studies to test the activity of fusion proteinsand polynucleotides of the invention (e.g., gene therapy).

Example 29 Construction of GAS Reporter Construct

[0968] One signal transduction pathway involved in the differentiationand proliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

[0969] GAS and ISRE elements are recognized by a class of transcriptionfactors called Signal Transducers and Activators of Transcription, or“STATs.” There are six members of the STATs family. Stat1 and Stat3 arepresent in many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class 1, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

[0970] The STATs are activated to translocate from the cytoplasm to thenucleus upon tyrosine phosphorylation by a set of kinases known as theJanus Kinase (“Jaks”) family. Jaks represent a distinct family ofsoluble tyrosine kinases and include Tyk2, Jak 1, Jak2, and Jak3. Thesekinases display significant sequence similarity and are generallycatalytically inactive in resting cells.

[0971] The Jaks are activated by a wide range of receptors summarized inthe Table below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995)). A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xaa-Trp-Ser (SEQ ID NO:37)).

[0972] Thus, on binding of a ligand to a receptor, Jaks are activated,which in turn activate STATs, which then translocate and bind to GASelements. This entire process is encompassed in the Jaks-STATs signaltransduction pathway. Therefore, activation of the Jaks-STATs pathway,reflected by the binding of the GAS or the ISRE element, can be used toindicate proteins involved in the proliferation and differentiation ofcells. For example, growth factors and cytokines are known to activatethe Jaks-STATs pathway (See Table below). Thus, by using GAS elementslinked to reporter molecules, activators of the Jaks-STATs pathway canbe identified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) orISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 >Lys6 > IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ?1, 3 GAS (IRF1 > Lys6 > IFP) Il-11(Pleiotropic) ? + ? ? 1, 3OnM(Pleiotropic) ? + + ? 1, 3 LIF(Pleiotropic) ? + + ? 1, 3CNTF(Pleiotropic) −/+ + + ? 1, 3 G-CSF(Pleiotropic) ? + ? ? 1, 3IL-12(Pleiotropic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1,3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS(IRF1 = IFP >> Ly6)(IgH) IL-7(lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13(lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3(myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) − − + − 5 GASGM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ?+/− + − 1, 3, 5 EPO ? − + − 5 GAS(B-CAS > IRF1 = IFP >> Ly6) ReceptorTyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1? + + − 1, 3 GAS (not IRF1)

[0973] To construct a synthetic GAS containing promoter element, whichis used in the Biological Assays described in Examples 32-33, a PCRbased strategy is employed to generate a GAS-SV40 promoter sequence. The5′ primer contains four tandem copies of the GAS binding site found inthe IRFI promoter and previously demonstrated to bind STATs uponinduction with a range of cytokines (Rothman et al., Immunity 1:457-468(1994).), although other GAS or ISRE elements can be used instead. The5′ primer also contains 18 bp of sequence complementary to the SV40early promoter sequence and is flanked with an XhoI site. The sequenceof the 5′ primer is: (SEQ ID NO:38)5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

[0974] The downstream primer is complementary to the SV40 promoter andis flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQID NO:39) PCR amplification is performed using the SV40 promotertemplate present in the B-gal:promoter plasmid obtained from Clontech.The resulting PCR fragment is digested with XhoI/Hind III and subclonedinto BLSK2-. (Stratagene.) Sequencing with forward and reverse primersconfirms that the insert contains the following sequence: (SEQ ID NO:40)5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

[0975] With this GAS promoter element linked to the SV40 promoter, aGAS:SEAP2 reporter construct is next engineered. Here, the reportermolecule is a secreted alkaline phosphatase, or “SEAP.” Clearly,however, any reporter molecule can be instead of SEAP, in this or in anyof the other Examples. Well known reporter molecules that can be usedinstead of SEAP include chloramphenicol acetyltransferase (CAT),luciferase, alkaline phosphatase, B-galactosidase, green fluorescentprotein (GFP), or any protein detectable by an antibody.

[0976] The above sequence confirmed synthetic GAS-SV40 promoter elementis subcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

[0977] Thus, in order to generate mammalian stable cell lines expressingthe GAS-SEAP reporter, the GAS-SEAP cassette is removed from theGAS-SEAP vector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 32-33.

[0978] Other constructs can be made using the above description andreplacing GAS with a different promoter sequence. For example,construction of reporter molecules containing EGR and NF-KB promotersequences are described in Examples 34 and 35. However, many otherpromoters can be substituted using the protocols described in theseExamples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can besubstituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB,11-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used totest reporter construct activity, such as HELA (epithelial), HUVEC(endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), orCardiomyocyte.

Example 30 Assay for SEAP Activity

[0979] As a reporter molecule for the assays described in examplesdisclosed herein, SEAP activity is assayed using the TropixPhospho-light Kit (Cat. BP-400) according to the following generalprocedure. The Tropix Phospho-light Kit supplies the Dilution, Assay,and Reaction Buffers used below.

[0980] Prime a dispenser with the 2.5×Dilution Buffer and dispense 15 ulof 2.5×dilution buffer into Optiplates containing 35 ul of a solutioncontaining an albumin fusion protein of the invention. Seal the plateswith a plastic sealer and incubate at 65 degree C. for 30 min. Separatethe Optiplates to avoid uneven heating.

[0981] Cool the samples to room temperature for 15 minutes. Empty thedispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer andincubate at room temperature 5 min. Empty the dispenser and prime withthe Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer andincubate at room temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on a luminometer, thus one should treat 5 plates ateach time and start the second set 10 minutes later.

[0982] Read the relative light unit in the luminometer. Set H12 asblank, and print the results. An increase in chemiluminescence indicatesreporter activity.

[0983] Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml)CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 1690 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 623 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 1859.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 21510.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 24512.25 48 250 12.5 49 255 12.75 50 260 13

Example 31 Assay Identifying Neuronal Activity

[0984] When cells undergo differentiation and proliferation, a group ofgenes are activated through many different signal transduction pathways.One of these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, the ability of fusion proteins of the invention toactivate cells can be assessed.

[0985] Particularly, the following protocol is used to assess neuronalactivity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells)are known to proliferate and/or differentiate by activation with anumber of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF(nerve growth factor), and EGF (epidermal growth factor). The EGR1 geneexpression is activated during this treatment. Thus, by stablytransfecting PC12 cells with a construct containing an EGR promoterlinked to SEAP reporter, activation of PC12 cells by an albumin fusionprotein of the present invention can be assessed.

[0986] The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: (SEQ ID NO:41)5′GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:42)5′GCGAAGCTTCGCGACTCCCCGGATCCGCTC-3′

[0987] Using the GAS:SEAP/Neo vector produced in Example 29, EGR1amplified product can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

[0988] To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

[0989] PC12 cells are routinely grown in RPMI-1640 medium (BioWhittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat.#12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplementedwith 100 units/ml penicillin and 100 ug,/ml streptomycin on a precoated10 cm tissue culture dish. One to four split is done every three to fourdays. Cells are removed from the plates by scraping and resuspended withpipetting up and down for more than 15 times.

[0990] Transfect the EGR/SEAP/Neo construct into PC12 using techniquesknown in the art. EGR-SEAP/PC12 stable cells are obtained by growing thecells in 300 ug/ml G418. The G418-free medium is used for routine growthbut every one to two months, the cells should be re-grown in 300 ug/mlG418 for couple of passages.

[0991] To assay for neuronal activity, a 10 cm plate with cells around70 to 80% confluent is screened by removing the old medium. Wash thecells once with PBS (Phosphate buffered saline). Then starve the cellsin low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBSwith antibiotics) overnight.

[0992] The next morning, remove the medium and wash the cells with PBS.Scrape off the cells from the plate, suspend the cells well in 2 ml lowserum medium. Count the cell number and add more low serum medium toreach final cell density as 5×10⁵ cells/ml.

[0993] Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add a series of differentconcentrations of an albumin fusion protein of the inventon, 37 degreeC. for 48 to 72 hr. As a positive control, a growth factor known toactivate PC12 cells through EGR can be used, such as 50 μg/ul ofNeuronal Growth Factor (NGF). Over fifty-fold induction of SEAP istypically seen in the positive control wells. SEAP assay may beroutinely performed using techniques known in the art and/or asdescribed in Example 30.

Example 32 Assay for T-Cell Activity

[0994] The following protocol is used to assess T-cell activity byidentifying factors, and determining whether an albumin fusion proteinof the invention proliferates and/or differentiates T-cells. T-cellactivity is assessed using the GAS/SEAP/Neo construct produced inExample 29. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. TheT-cell used in this assay is Jurkat T-cells (ATCC Accession No.TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4cells (ATCC Accession No. CRL-1582) cells can also be used.

[0995] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In orderto generate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

[0996] Specifically, the following protocol will yield sufficient cellsfor 75 wells containing 200 ul of cells. Thus, it is either scaled up,or performed in multiple to generate sufficient cells for multiple 96well plates. Jurkat cells are maintained in RPMI+10% serum with1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ugof plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul ofDMRIE-C and incubate at room temperature for 15-45 mins.

[0997] During the incubation period, count cell concentration, spin downthe required number of cells (107 per transfection), and resuspend inOPTI-MEM to a final concentration of 107 cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

[0998] The Jurkat:GAS-SEAP stable reporter lines are maintained inRPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells aretreated with varying concentrations of one or more fusion proteins ofthe present invention.

[0999] On the day of treatment with the fusion protein, the cells shouldbe washed and resuspended in fresh RPMI+10% serum to a density of500,000 cells per ml. The exact number of cells required will depend onthe number of fusion proteins and the number of different concentrationsof fusion proteins being screened. For one 96 well plate, approximately10 million cells (for 10 plates, 100 million cells) are required.

[1000] The well dishes containing Jurkat cells treated with the fusionprotein are placed in an incubator for 48 hrs (note: this time isvariable between 48-72 hrs). 35 ul samples from each well are thentransferred to an opaque 96 well plate using a 12 channel pipette. Theopaque plates should be covered (using sellophene covers) and stored at−20 degree C. until SEAP assays are performed according to Example 30.The plates containing the remaining treated cells are placed at 4 degreeC. and serve as a source of material for repeating the assay on aspecific well if desired.

[1001] As a positive control, 100 Unit/ml interferon gamma can be usedwhich is known to activate Jurkat T cells. Over 30 fold induction istypically observed in the positive control wells.

[1002] The above protocol may be used in the generation of bothtransient, as well as, stable transfected cells, which would be apparentto those of skill in the art.

Example 33 Assay for T-Cell Activity

[1003] NF-KB (Nuclear Factor KB) is a transcription factor activated bya wide variety of agents including the inflammatory cytokines IL-1 andTNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposureto LPS or thrombin, and by expression of certain viral gene products. Asa transcription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

[1004] In non-stimulated conditions, NF-KB is retained in the cytoplasmwith I-KB (Inhibitor KB). However, upon stimulation, I-KB isphosphorylated and degraded, causing NF-KB to shuttle to the nucleus,thereby activating transcription of target genes. Target genes activatedby NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

[1005] Due to its central role and ability to respond to a range ofstimuli, reporter constructs utilizing the NF-KB promoter element areused to screen the fusion protein. Activators or inhibitors of NF-KBwould be useful in treating, preventing, and/or diagnosing diseases. Forexample, inhibitors of NF-KB could be used to treat those diseasesrelated to the acute or chronic activation of NF-KB, such as rheumatoidarthritis.

[1006] To construct a vector containing the NF-KB promoter element, aPCR based strategy is employed. The upstream primer contains four tandemcopies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:43), 18 bp ofsequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site: (SEQ ID NO:44)5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGAGTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′

[1007] The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:39)

[1008] PCR amplification is performed using the SV40 promoter templatepresent in the pB-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI and Hind III and subclonedinto BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirmsthe insert contains the following sequence: (SEQ ID NO:45)5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3′

[1009] Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

[1010] In order to generate stable mammalian cell lines, theNF-KBISV40/SEAP cassette is removed from the above NF-KB/SEAP vectorusing restriction enzymes SalI and NotI, and inserted into a vectorcontaining neomycin resistance. Particularly, the NF-KB/SV40/SEAPcassette was inserted into pGFP-1 (Clontech), replacing the GFP gene,after restricting pGFP-1 with SalI and NotI.

[1011] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 32. Similarly, the method for assaying fusion proteins withthese stable Jurkat T-cells is also described in Example 32. As apositive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wellsH9, H10, and HI1, with a 5-10 fold activation typically observed.

Example 33 Assay Identifying Myeloid Activity

[1012] The following protocol is used to assess myeloid activity of analbumin fusion protein of the present invention by determining whetherthe fusion protein proliferates and/or differentiates myeloid cells.Myeloid cell activity is assessed using the GAS/SEAP/Neo constructproduced in Example 29. Thus, factors that increase SEAP activityindicate the ability to activate the Jaks-STATS signal transductionpathway. The myeloid cell used in this assay is U937, a pre-monocytecell line, although TF-1, HL60, or KG1 can be used.

[1013] To transiently transfect U937 cells with the GAS/SEAP/Neoconstruct produced in Example 29, a DEAE-Dextran method (Kharbanda et.al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First,harvest 2×10⁷ U937 cells and wash with PBS. The U937 cells are usuallygrown in RPMI 1640 medium containing 10% heat-inactivated fetal bovineserum (FBS) supplemented with 100 units/ml penicillin and 100 mg/mlstreptomycin.

[1014] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄₀.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

[1015] Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

[1016] The GAS-SEAP/U937 stable cells are obtained by growing the cellsin 400 ug/ml G418. The G418-free medium is used for routine growth butevery one to two months, the cells should be re-grown in 400 ug/ml G418for couple of passages.

[1017] These cells are tested by harvesting 1×10⁸ cells (this is enoughfor ten 96-well plates assay) and wash with PBS. Suspend the cells in200 ml above described growth medium, with a final density of 5×10⁵cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵cells/well).

[1018] Add different concentrations of the fusion protein. Incubate at37 degee C. for 48 to 72 hr. As a positive control, 100 Unit/mlinterferon gamma can be used which is known to activate U937 cells. Over30 fold induction is typically observed in the positive control wells.SEAP assay the supernatant according to methods known in the art and/orthe protocol described in Example 30.

Example 34 Assay Identifying Changes in Small Molecule Concentration andMembrane Permeability

[1019] Binding of a ligand to a receptor is known to alter intracellularlevels of small molecules, such as calcium, potassium, sodium, and pH,as well as alter membrane potential. These alterations can be measuredin an assay to identify fusion proteins which bind to receptors of aparticular cell. Although the following protocol describes an assay forcalcium, this protocol can easily be modified to detect changes inpotassium, sodium, pH, membrane potential, or any other small moleculewhich is detectable by a fluorescent probe.

[1020] The following assay uses Fluorometric Imaging Plate Reader(“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes)that bind small molecules. Clearly, any fluorescent molecule detecting asmall molecule can be used instead of the calcium fluorescent molecule,fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

[1021] For adherent cells, seed the cells at 10,000-20,000 cells/well ina Co-star black 96-well plate with clear bottom. The plate is incubatedin a CO₂ incubator for 20 hours. The adherent cells are washed two timesin Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

[1022] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acidDMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is addedto each well. The plate is incubated at 37 degrees C. in a CO₂ incubatorfor 60 min. The plate is washed four times in the Biotek washer withHBSS leaving 100 ul of buffer.

[1023] For non-adherent cells, the cells are spun down from culturemedia. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-mlconical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSOis added to each ml of cell suspension. The tube is then placed in a 37degrees C. water bath for 30-60 min. The cells are washed twice withHBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate,100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plateis then washed once in Denley Cell Wash with 200 ul, followed by anaspiration step to 100 ul final volume.

[1024] For a non-cell based assay, each well contains a fluorescentmolecule, such as fluo-4. The fusion protein of the invention is addedto the well, and a change in fluorescence is detected.

[1025] To measure the fluorescence of intracellular calcium, the FLIPRis set for the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by an albumin fusion protein of the present invention or amolecule induced by an albumin fusion protein of the present invention,which has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 35 Assay Identifying Tyrosine Kinase Activity

[1026] The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase (RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

[1027] Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, Ick, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

[1028] Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether an albumin fusion proteinof the present invention or a molecule induced by a fusion proetin ofthe present invention is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

[1029] Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

[1030] To prepare extracts, A431 cells are seeded onto the nylonmembranes of Loprodyne plates (20,000/200 ml/well) and culturedovernight in complete medium. Cells are quiesced by incubation inserum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF(60 ng/ml) or a different concentrations of an albumin fusion protein ofthe invention, the medium was removed and 100 ml of extraction buffer((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mMNa3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors (#1836170)obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added toeach well and the plate is shaken on a rotating shaker for 5 minutes at4° C. The plate is then placed in a vacuum transfer manifold and theextract filtered through the 0.45 mm membrane bottoms of each well usinghouse vacuum. Extracts are collected in a 96-well catch/assay plate inthe bottom of the vacuum manifold and immediately placed on ice. Toobtain extracts clarified by centrifugation, the content of each well,after detergent solubilization for 5 minutes, is removed and centrifugedfor 15 minutes at 4 degree C. at 16,000×g.

[1031] Test the filtered extracts for levels of tyrosine kinaseactivity. Although many methods of detecting tyrosine kinase activityare known, one method is described here.

[1032] Generally, the tyrosine kinase activity of an albumin fusionprotein of the invention is evaluated by determining its ability tophosphorylate a tyrosine residue on a specific substrate (a biotinylatedpeptide). Biotinylated peptides that can be used for this purposeinclude PSK1 (corresponding to amino acids 6-20 of the cell divisionkinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 ofgastrin). Both peptides are substrates for a range of tyrosine kinasesand are available from Boehringer Mannheim.

[1033] The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5×Assay Buffer (40mM imidazole hydrochloride, pH 7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then Sul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

[1034] The tyrosine kinase assay reaction is then terminated by adding10 ul of 120 mm EDTA and place the reactions on ice.

[1035] Tyrosine kinase activity is determined by transferring 50 ulaliquot of reaction mixture to a microtiter plate (MTP) module andincubating at 37 degree C. for 20 min. This allows the streptavidincoated 96 well plate to associate with the biotinylated peptide. Washthe MTP module with 300 ul/well of PBS four times. Next add 75 ul ofanti-phospolyrosine antibody conjugated to horse radishperoxidase(anti-P-Tyr-POD(0.5u/ml)) to each well and incubate at 37degree C. for one hour. Wash the well as above.

[1036] Next add 100 ul of peroxidase substrate solution (BoehringerMannheim) and incubate at room temperature for at least 5 mins (up to 30min). Measure the absorbance of the sample at 405 nm by using ELISAreader. The level of bound peroxidase activity is quantitated using anELISA reader and reflects the level of tyrosine kinase activity.

Example 36 Assay Identifying Phosphorylation Activity

[1037] As a potential alternative and/or complement to the assay ofprotein tyrosine kinase activity described in Example 35, an assay whichdetects activation (phosphorylation) of major intracellular signaltransduction intermediates can also be used. For example, as describedbelow one particular assay can detect tyrosine phosphorylation of theErk-1 and Erk-2 kinases. However, phosphorylation of other molecules,such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src,Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as anyother phosphoserine, phosphotyrosine, or phosphothreonine molecule, canbe detected by substituting these molecules for Erk-1 or Erk-2 in thefollowing assay.

[1038] Specifically, assay plates are made by coating the wells of a96-well ELISA plate with 0.1 ml of protein G (lug/ml) for 2 hr at roomtemp, (RT). The plates are then rinsed with PBS and blocked with 3%BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2commercial monoclonal antibodies (10 ng/well) against Erk-1 and Erk-2 (1hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, thisstep can easily be modified by substituting a monoclonal antibodydetecting any of the above described molecules.) After 3-5 rinses withPBS, the plates are stored at 4 degree C. until use.

[1039] A431 cells are seeded at 20,000/well in a 96-well Loprodynefilterplate and cultured overnight in growth medium. The cells are thenstarved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or varying concentrations of the fusion protein of theinvention for 5-20 minutes. The cells are then solubilized and extractsfiltered directly into the assay plate.

[1040] After incubation with the extract for 1 hr at RT, the wells areagain rinsed. As a positive control, a commercial preparation of MAPkinase (10 ng/well) is used in place of A431 extract. Plates are thentreated with a commercial polyclonal (rabbit) antibody (lug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by the fusion protein of thepresent invention or a molecule induced by an albumin fusion protein ofthe present invention.

Example 37 Assay for the Stimulation of Bone Marrow CD34+ CellProliferation

[1041] This assay is based on the ability of human CD34+ to proliferatein the presence of hematopoietic growth factors and evaluates theability of fusion proteins of the inventon to stimulate proliferation ofCD34+ cells.

[1042] It has been previously shown that most mature precursors willrespond to only a single signal. More immature precursors require atleast two signals to respond. Therefore, to test the effect of fusionproteins of the invention on hematopoietic activity of a wide range ofprogenitor cells, the assay contains a given fusion protein of theinvention in the presence or absence of hematopoietic growth factors.Isolated cells are cultured for 5 days in the presence of Stem CellFactor (SCF) in combination with tested sample. SCF alone has a verylimited effect on the proliferation of bone marrow (BM) cells, acting insuch conditions only as a “survival” factor. However, combined with anyfactor exhibiting stimulatory effect on these cells (e.g., IL-3), SCFwill cause a synergistic effect. Therefore, if the tested fusion proteinhas a stimulatory effect on hematopoietic progenitors, such activity canbe easily detected. Since normal BM cells have a low level of cyclingcells, it is likely that any inhibitory effect of a given fusion proteinmight not be detected. Accordingly, assays for an inhibitory effect onprogenitors is preferably tested in cells that are first subjected to invitro stimulation with SCF+IL+3, and then contacted with the compoundthat is being evaluated for inhibition of such induced proliferation.

[1043] Briefly. CD34+ cells are isolated using methods known in the art.The cells are thawed and resuspended in medium (QBSF 60 serum-freemedium with 1% L-glutamine (500 ml) Quality Biological, Inc.,Gaithersburg, Md. Cat#160-20101). After several gentle centrifugationsteps at 200×g, cells are allowed to rest for one hour. The cell countis adjusted to 2.5×10⁵ cells/ml. During this time, 100 μl of sterilewater is added to the peripheral wells of a 96-well plate. The cytokinesthat can be tested with an albumin fusion protein of the invention inthis assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at 50ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems,Minneapolis, Minn., Cat# 203-ML) at 30 ng/ml. After one hour, 10 μl ofprepared cytokines, varying concentrations of an albumin fusion proteinof the invention, and 20 μl of diluted cells are added to the mediawhich is already present in the wells to allow for a final total volumeof 100 μl. The plates are then placed in a 37° C./5% CO₂ incubator forfive days.

[1044] Eighteen hours before the assay is harvested, 0.5 μCi/well of[3H] Thymidine is added in a 10 μl volume to each well to determine theproliferation rate. The experiment is terminated by harvesting the cellsfrom each 96-well plate to a filtermat using the Tomtec Harvester 96.After harvesting, the filtermats are dried, trimmed and placed intoOmniFilter assemblies consisting of one OmniFilter plate and oneOmniFilter Tray. 60 μl Microscint is added to each well and the platesealed with TopSeal-A press-on sealing film A bar code 15 sticker isaffixed to the first plate for counting. The sealed plates are thenloaded and the level of radioactivity determined via the Packard TopCount and the printed data collected for analysis. The level ofradioactivity reflects the amount of cell proliferation.

[1045] The studies described in this example test the activity of agiven fusion protein to stimulate bone marrow CD34+ cell proliferation.One skilled in the art could easily modify the exemplified studies totest the activity of fusion porteins and polynucleotides of theinvention (e.g. gene therapy) as well as agonists and antagoniststhereof. The ability of an albumin fusion protein of the invention tostimulate the proliferation of bone marrow CD34+ cells indicates thatthe albumin fusion protein and/or polynucleotides corresponding to thefusion protein are useful for the diagnosis and treatment of disordersaffecting the immune system and hematopoiesis. Representative uses aredescribed in the “Immune Activity” and “Infectious Disease” sectionsabove, and elsewhere herein.

Example 38 Assay for Extracellular Matrix Enhanced Cell Response (EMECR)

[1046] The objective of the Extracellular Matrix Enhanced Cell Response(EMECR) assay is to evaluate the ability of fusion proteins of theinvention to act on hematopoietic stem cells in the context of theextracellular matrix (ECM) induced signal.

[1047] Cells respond to the regulatory factors in the context ofsignal(s) received from the surrounding microenvironment. For example,fibroblasts, and endothelial and epithelial stem cells fail to replicatein the absence of signals from the ECM. Hematopoietic stem cells canundergo self-renewal in the bone marrow, but not in in vitro suspensionculture. The ability of stem cells to undergo self-renewal in vitro isdependent upon their interaction with the stromal cells and the ECMprotein fibronectin (fn). Adhesion of cells to fn is mediated by theα₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human andmouse hematopoietic stem cells. The factor(s) which integrate with theECM environment and are responsible for stimulating stem cellself-renewal havea not yet been identified. Discovery of such factorsshould be of great interest in gene therapy and bone marrow transplantapplications Briefly, polystyrene, non tissue culture treated, 96-wellplates are coated with fn fragment at a coating concentration of 0.2μg/cm². Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 mlof serum-free medium. Cells cultured in the presence of IL-3 (5ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditionsunder which little self-renewal but pronounced differentiation of thestem cells is to be expected. Albumin fusion proteins of the inventionare tested with appropriate negative controls in the presence andabsence of SCF(5.0 ng/ml), where volume of the administed compositioncontaining the albumin fusion protein of the invention represents 10% ofthe total assay volume. The plated cells are then allowed to grow byincubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂)tissue culture incubator for 7 days. The number of proliferating cellswithin the wells is then quantitated by measuring thymidineincorporation into cellular DNA. Verification of the positive hits inthe assay will require phenotypic characterization of the cells, whichcan be accomplished by scaling up of the culture system and usingappropriate antibody reagents against cell surface antigens and FACScan.

[1048] One skilled in the art could easily modify the exemplifiedstudies to test the activity of albumin fusion proteins andpolynucleotides of the invention (e.g., gene therapy).

[1049] If a particular fusion protein of the present invention is foundto be a stimulator of hematopoietic progenitors, the fusion protein andpolynucleotides corresponding to the fusion protein may be useful forexample, in the diagnosis and treatment of disorders affecting theimmune system and hematopoiesis. Representative uses are described inthe “Immune Activity” and “Infectious Disease” sections above, andelsewhere herein. The fusion protein may also be useful in the expansionof stem cells and committed progenitors of various blood lineages, andin the differentiation and/or proliferation of various cell types.

[1050] Additionally, the albumin fusion proteins of the invention andpolynucleotides encoding albumin fusion proteins of the invention, mayalso be employed to inhibit the proliferation and differentiation ofhematopoietic cells and therefore may be employed to protect bone marrowstem cells from chemotherapeutic agents during chemotherapy. Thisantiproliferative effect may allow administration of higher doses ofchemotherapeutic agents and, therefore, more effective chemotherapeutictreatment.

[1051] Moreover, fusion proteins of the invention and polynucleotidesencoding albumin fusion proteins of the invention may also be useful forthe treatment and diagnosis of hematopoietic related disorders such as,anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, sincestromal cells are important in the production of cells of hematopoieticlineages. The uses include bone marrow cell ex-vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia.

Example 39 Human Dermal Fibroblast and Aortic Smooth Muscle CellProliferation

[1052] An albumin fusion protein of the invention is added to culturesof normal human dermal fibroblasts (NHDF) and human aortic smooth musclecells (AoSMC) and two co-assays are performed with each sample. Thefirst assay examines the effect of the fusion protein on theproliferation of normal human dermal fibroblasts (NHDF) or aortic smoothmuscle cells (AoSMC). Aberrant growth of fibroblasts or smooth musclecells is a part of several pathological processes, including fibrosis,and restenosis. The second assay examines IL6 production by both NHDFand SMC. IL6 production is an indication of functional activation.Activated cells will have increased production of a number of cytokinesand other factors, which can result in a proinflammatory orimmunomodulatory outcome. Assays are run with and without co-TNFastimulation, in order to check for costimulatory or inhibitory activity.

[1053] Briefly, on day 1, 96-well black plates are set up with 1000cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media.NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5mg/ml insulin, 50 mg/ml gentamycin, 2%FBS, while AoSMC culture mediacontains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5%FBS. Afterincubation at 37° C. for at least 4-5 hours culture media is aspiratedand replaced with growth arrest media. Growth arrest media for NHDFcontains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, whilegrowth arrest media for AoSMC contains SM basal media, 50 mg/mlgentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37° C. untilday 2.

[1054] On day 2, serial dilutions and templates of an albumin fusionprotein of the invention are designed such that they always includemedia controls and known-protein controls. For both stimulation andinhibition experiments, proteins are diluted in growth arrest media. Forinhibition experiments, TNFa is added to a final concentration of 2ng/ml (NHDF) or Sng/ml (AoSMC). Add ⅓ vol media containing controls oran albumin fusion protein of the invention and incubate at 37 degreesC./5% CO₂ until day 5.

[1055] Transfer 60 μl from each well to another labeled 96-well plate,cover with a plate-sealer, and store at 4 degrees C. until Day 6 (forIL6 ELISA). To the remaining 100 μl in the cell culture plate,aseptically add Alamar Blue in an amount equal to 10% of the culturevolume (10%1). Return plates to incubator for 3 to 4 hours. Then measurefluorescence with excitation at 530 nm and emission at 590 nm using theCytoFluor. This yields the growth stimulation/inhibition data.

[1056] On day 5, the IL6 ELISA is performed by coating a 96 well platewith 50-100 ul/well of Anti-Human 11L6 Monoclonal antibody diluted inPBS, pH 7.4, incubate ON at room temperature.

[1057] On day 6, empty the plates into the sink and blot on papertowels. Prepare Assay Buffer containing PBS with 4% BSA. Block theplates with 200 μl/well of Pierce Super Block blocking buffer in PBS for1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blotplates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions ofIL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samplesto top row of plate. Cover the plates and incubate for 2 hours at RT onshaker.

[1058] Plates are washed with wash buffer and blotted on paper towels.Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100μl/well. Cover the plate and incubate 1 h at RT. Plates are again washedwith wash buffer and blotted on paper towels.

[1059] Add 100 μl/well of Enhancement Solution. Shake for 5 minutes.Read the plate on the Wallac DELFIA Fluorometer. Readings fromtriplicate samples in each assay were tabulated and averaged.

[1060] A positive result in this assay suggests AoSMC cell proliferationand that the albumin fusion protein may be involved in dermal fibroblastproliferation and/or smooth muscle cell proliferation. A positive resultalso suggests many potential uses of the fusion protein andpolynucleotides encoding the albumin fusion protein. For example,inflammation and immune responses, wound healing, and angiogenesis, asdetailed throughout this specification. Particularly, fusion proteinsmay be used in wound healing and dermal regeneration, as well as thepromotion of vasculogenesis, both of the blood vessels and lymphatics.The growth of vessels can be used in the treatment of, for example,cardiovascular diseases. Additionally, fusion proteins showingantagonistic activity in this assay may be useful in treating diseases,disorders, and/or conditions which involve angiogenesis by acting as ananti-vascular agent (e.g., anti-angiogenesis). These diseases,disorders, and/or conditions are known in the art and/or are describedherein, such as, for example, malignancies, solid tumors, benign tumors,for example hemangiomas, acoustic neuromas, neurofibromas, trachomas,and pyogenic granulomas; artheroscleric plaques; ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia(abnormal blood vessel growth) of the eye; rheumatoid arthritis;psoriasis; delayed wound healing; endometriosis; vasculogenesis;granulations; hypertrophic scars (keloids); nonunion fractures;scleroderma; trachoma; vascular adhesions; myocardial angiogenesis;coronary collaterals; cerebral collaterals; arterioyenous malformations;ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis. Moreover, albumin fusion proteins that act asantagonists in this assay may be useful in treatinganti-hyperproliferative diseases and/or anti-inflammatory known in theart and/or described herein.

Example 40 Cellular Adhesion Molecule (CAM) Expression on EndothelialCells

[1061] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[1062] Briefly, endothelial cells (e.g., Human Umbilical VeinEndothelial cells (HUVECs)) are grown in a standard 96 well plate toconfluence, growth medium is removed from the cells and replaced with100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing(containing an albumin fusion protein of the invention) and positive ornegative controls are added to the plate in triplicate (in 10 μlvolumes). Plates are then incubated at 37° C. for either 5 h (selectinand integrin expression) or 24 h (integrin expression only). Plates areaspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(withCa++ and Mg++) is added to each well. Plates are held at 4° C. for 30min. Fixative is removed from the wells and wells are washed 1×withPBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody isadded to the test and control wells. Anti-ICAM-1-Biotin,Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at aconcentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody).Cells are incubated at 37° C. for 30 min. in a humidified environment.Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 μl of dilutedExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein asthe working dilution) are added to each well and incubated at 37° C. for30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH10.4). 100 μl of pNPP substrate in glycine buffer is added to each testwell. Standard wells in triplicate are prepared from the workingdilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer:1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5).5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to eachof the standard wells. The plate is incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The plate is read on aplate reader at 405 nm using the background subtraction option on blankwells filled with glycine buffer only. Additionally, the template is setup to indicate the concentration of AP-conjugate in each standard well[5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount ofbound AP-conjugate in each sample.

Example 41 Alamar Blue Endothelial Cells Proliferation Assay

[1063] This assay may be used to quantitatively determine proteinmediated inhibition of bFGF-induced proliferation of Bovine LymphaticEndothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) orHuman Microvascular Uterine Myometrial Cells (UTMECs). This assayincorporates a fluorometric growth indicator based on detection ofmetabolic activity. A standard Alamar Blue Proliferation Assay isprepared in EGM-2MV with 10 ng/ml of bFGF added as a source ofendothelial cell stimulation. This assay may be used with a variety ofendothelial cells with slight changes in growth medium and cellconcentration. Dilutions of protein batches to be tested are diluted asappropriate. Serum-free medium (GIBCO SFM) without bFGF is used as anon-stimulated control and Angiostatin or TSP-1 are included as a knowninhibitory controls.

[1064] Briefly, LEC, BAECs or UTMECs are seeded in growth media at adensity of 5000 to 2000 cells/well in a 96 well plate and placed at 37degrees C. overnight. After the overnight incubation of the cells, thegrowth media is removed and replaced with GIBCO EC-SFM. The cells aretreated with the appropriate dilutions of an albumin fusion protein ofthe invention or control protein sample(s) (prepared in SFM) intriplicate wells with additional bFGF to a concentration of 10 ng/ml.Once the cells have been treated with the samples, the plate(s) is/areplaced back in the 37° C. incubator for three days. After three days 10ml of stock alamar blue (Biosource Cat#DAL1100) is added to each welland the plate(s) is/are placed back in the 37° C. incubator for fourhours. The plate(s) are then read at 530 nm excitation and 590 nmemission using the CytoFluor fluorescence reader. Direct output isrecorded in relative fluorescence units.

[1065] Alamar blue is an oxidation-reduction indicator that bothfluoresces and changes color in response to chemical reduction of growthmedium resulting from cell growth. As cells grow in culture, innatemetabolic activity results in a chemical reduction of the immediatesurrounding environment. Reduction related to growth causes theindicator to change from oxidized (non-fluorescent blue) form to reduced(fluorescent red) form (i.e., stimulated proliferation will produce astronger signal and inhibited proliferation will produce a weaker signaland the total signal is proportional to the total number of cells aswell as their metabolic activity). The background level of activity isobserved with the starvation medium alone. This is compared to theoutput observed from the positive control samples (bFGF in growthmedium) and protein dilutions.

Example 42 Detection of Inhibition of a Mixed Lymphocyte Reaction

[1066] This assay can be used to detect and evaluate inhibition of aMixed Lymphocyte Reaction (MLR) by fusion proteins of the invention.Inhibition of a MLR may be due to a direct effect on cell proliferationand viability, modulation of costimulatory molecules on interactingcells, modulation of adhesiveness between lymphocytes and accessorycells, or modulation of cytokine production by accessory cells. Multiplecells may be targeted by the albumin fusion proteins that inhibit MLRsince the peripheral blood mononuclear fraction used in this assayincludes T, B and natural killer lymphocytes, as well as monocytes anddendritic cells.

[1067] Albumin fusion proteins of the invention found to inhibit the MLRmay find application in diseases associated with lymphocyte and monocyteactivation or proliferation. These include, but are not limited to,diseases such as asthma, arthritis, diabetes, inflammatory skinconditions, psoriasis, eczema, systemic lupus erythematosus, multiplesclerosis, glomerulonephritis, inflammatory bowel disease, crohn'sdisease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. hostdisease, host vs. graft disease, hepatitis, leukemia and lymphoma.

[1068] Briefly, PBMCs from human donors are purified by density gradientcentrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from twodonors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies,Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCsfrom a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters ofPBMCs from each donor is added to wells of a 96-well round bottommicrotiter plate. Dilutions of the fusion protein test material (50 μl)is added in triplicate to microtiter wells. Test samples (of the proteinof interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems,Minneapolis, Minn., catalog number 202-IL) is added to a finalconcentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11,catalog number MAB379) is added to a final concentration of 10 μg/ml.Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H]thymidine is added to wells for the last 16 hrs of culture. Cells areharvested and thymidine incorporation determined using a PackardTopCount. Data is expressed as the mean and standard deviation oftriplicate determinations.

[1069] Samples of the fusion protein of interest are screened inseparate experiments and compared to the negative control treatment,anti-CD4 mAb, which inhibits proliferation of lymphocytes and thepositive control treatment, IL-2 (either as recombinant material orsupernatant), which enhances proliferation of lymphocytes.

Example 43 Assays for Protease Activity

[1070] The following assay may be used to assess protease activity of analbumin fusion protein of the invention.

[1071] Gelatin and casein zymography are performed essentially asdescribed (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson etal., Jounal of Urology, 149:653-658 (1993)). Samples are run on 10%polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3at 37° C. 5 to 16 hours. After staining in amido black areas ofproteolysis apear as clear areas agains the blue-black background.Trypsin (Sigma T8642) is used as a positive control.

[1072] Protease activity is also determined by monitoring the cleavageof n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500. Reactionsare set up in (25 mMNaPO₄, 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samplesare added and the change in adsorbance at 260 nm is monitored on theBeckman DU-6 spectrophotometer in the time-drive mode. Trypsin is usedas a positive control.

[1073] Additional assays based upon the release of acid-soluble peptidesfrom casein or hemoglobin measured as adsorbance at 280 nm orcolorimetrically using the Folin method are performed as described inBergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assaysinvolve the solubilization of chromogenic substrates (Ward, AppliedScience, 251-317 (1983)).

Example 44 Identifying Serine Protease Substrate Specificity

[1074] Methods known in the art or described herein may be used todetermine the substrate specificity of the albumin fusion proteins ofthe present invention having serine protease activity. A preferredmethod of determining substrate specificity is by the use of positionalscanning synthetic combinatorial libraries as described in GB 2 324 529(incorporated herein in its entirety).

Example 45 Ligand Binding Assays

[1075] The following assay may be used to assess ligand binding activityof an albumin fusion protein of the invention.

[1076] Ligand binding assays provide a direct method for ascertainingreceptor pharmacology and are adaptable to a high throughput format. Thepurified ligand for an albumin fusion protein of the invention isradiolabeled to high specific activity (50-2000 Ci/mmol) for bindingstudies. A determination is then made that the process of radiolabelingdoes not diminish the activity of the ligand towards the fusion protein.Assay conditions for buffers, ions, pH and other modulators such asnucleotides are optimized to establish a workable signal to noise ratiofor both membrane and whole cell polypeptide sources. For these assays,specific polypeptide binding is defined as total associatedradioactivity minus the radioactivity measured in the presence of anexcess of unlabeled competing ligand. Where possible, more than onecompeting ligand is used to define residual nonspecific binding.

Example 46 Functional Assay in Xenopus Oocytes

[1077] Capped RNA transcripts from linearized plasmid templates encodingan albumin fusion protein of the invention is synthesized in vitro withRNA polymerases in accordance with standard procedures. In vitrotranscripts are suspended in water at a final concentration of 0.2mg/ml. Ovarian lobes are removed from adult female toads, Stage Vdefolliculated oocytes are obtained, and RNA transcripts (10 ng/oocytc)are injected in a 50 nl bolus using a microinjection apparatus. Twoelectrode voltage clamps are used to measure the currents fromindividual Xenopus oocytes in response fusion protein and polypeptideagonist exposure. Recordings are made in Ca2+ free Barth's medium atroom temperature. The Xenopus system can be used to screen known ligandsand tissue/cell extracts for activating ligands.

Example 47 Microphysiometric Assays

[1078] Activation of a wide variety of secondary messenger systemsresults in extrusion of small amounts of acid from a cell. The acidformed is largely as a result of the increased metabolic activityrequired to fuel the intracellular signaling process. The pH changes inthe media surrounding the cell are very small but are detectable by theCYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park,Calif.). The CYTOSENSOR is thus capable of detecting the ability of analbumin fusion protein of the invention to activate secondary messengersthat are coupled to an energy utilizing intracellular signaling pathway.

Example 48 Extract/Cell Supernatant Screening

[1079] A large number of mammalian receptors exist for which thereremains, as yet, no cognate activating ligand (agonist). Thus, activeligands for these receptors may not be included within the ligands banksas identified to date. Accordingly, the albumin fusion proteins of theinvention can also be functionally screened (using calcium, cAMP,microphysiometer, oocyte electrophysiology, etc., functional screens)against tissue extracts to identify natural ligands for the Therapeuticprotein portion and/or albumin protein portion of an albumin fusionprotein of the invention. Extracts that produce positive functionalresponses can be sequentially subfractionated until an activating ligandis isolated and identified.

Example 49 ATP-Binding Assay

[1080] The following assay may be used to assess ATP-binding activity offusion proteins of the invention.

[1081] ATP-binding activity of an albumin fusion protein of theinvention may be detected using the ATP-binding assay described in U.S.Pat. No. 5,858,719, which is herein incorporated by reference in itsentirety. Briefly, ATP-binding to an albumin fusion protein of theinvention is measured via photoaffinity labeling with 8-azido-ATP in acompetition assay. Reaction mixtures containing 1 mg/ml of ABC transportprotein are incubated with varying concentrations of ATP, or thenon-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at4° C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus8-azido-ATP (³²P-ATP) (5 mCi/[mol, ICN, Irvine Calif.) is added to afinal concentration of 100 μM and 0.5 ml aliquots are placed in thewells of a porcelain spot plate on ice. The plate is irradiated using ashort wave 254 nm UV lamp at a distance of 2.5 cm from the plate for twoone-minute intervals with a one-minute cooling interval in between. Thereaction is stopped by addition of dithiothreitol to a finalconcentration of 2 mM. The incubations are subjected to SDS-PAGEelectrophoresis, dried, and autoradiographed. Protein bandscorresponding to the albumin fusion proteins of the invention areexcised, and the radioactivity quantified. A decrease in radioactivitywith increasing ATP or adenly-5′-imidodiphosphate provides a measure ofATP affinity to the fusion protein.

Example 50 Phosphorylation Assay

[1082] In order to assay for phosphorylation activity of an albuminfusion protein of the invention, a phosphorylation assay as described inU.S. Pat. No. 5,958,405 (which is herein incorporated by reference) isutilized. Briefly, phosphorylation activity may be measured byphosphorylation of a protein substrate using gamma-labeled ³²P-ATP andquantitation of the incorporated radioactivity using a gammaradioisotope counter. The fusion portein of the invention is incubatedwith the protein substrate, ³²P-ATP, and a kinase buffer. The ³²Pincorporated into the substrate is then separated from free ³²P-ATP byelectrophoresis, and the incorporated ³²P is counted and compared to anegative control. Radioactivity counts above the negative control areindicative of phosphorylation activity of the fusion protein.

Example 51 Detection of Phosphorylation Activity (Activation) of anAlbumin Fusion Protein of the Invention in the Presence of PolypeptideLigands

[1083] Methods known in the art or described herein may be used todetermine the phosphorylation activity of an albumin fusion protein ofthe invention. A preferred method of determining phosphorylationactivity is by the use of the tyrosine phosphorylation assay asdescribed in U.S. Pat. No. 5,817,471 (incorporated herein by reference).

Example 52 Identification of Signal Transduction Proteins that Interactwith an Albumin Fusion Protein of the Present Invention

[1084] Albumin fusion proteins of the invention may serve as researchtools for the identification, characterization and purification ofsignal transduction pathway proteins or receptor proteins. Briefly, alabeled fusion protein of the invention is useful as a reagent for thepurification of molecules with which it interacts. In one embodiment ofaffinity purification, an albumin fusion protein of the invention iscovalently coupled to a chromatography column. Cell-free extract derivedfrom putative target cells, such as carcinoma tissues, is passed overthe column, and molecules with appropriate affinity bind to the albuminfusion protein. The protein complex is recovered from the column,dissociated, and the recovered molecule subjected to N-terminal proteinsequencing. This amino acid sequence is then used to identify thecaptured molecule or to design degenerate oligonucleotide probes forcloning the relevant gene from an appropriate cDNA library.

Example 53 IL-6 Bioassay

[1085] A variety of assays are known in the art for testing theproliferative effects of an albumin fusion protein of the invention. Forexample, one such asssay is the IL-6 Bioassay as described by Marz etal. (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is hereinincorporated by reference). After 68 hrs. at 37° C., the number ofviable cells is measured by adding the tetrazolium salt thiazolyl blue(MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysedby SDS and optical density is measured at 570 nm. Controls containingIL-6 (positive) and no cytokine (negative) are Briefly, IL-6 dependentB9 murine cells are washed three times in IL-6 free medium and plated ata concentration of 5,000 cells per well in 50 μl, and 50 μl of fusionprotein of the invention is added, utilized. Enhanced proliferation inthe test sample(s) (containing an albumin fusion protein of theinvention) relative to the negative control is indicative ofproliferative effects mediated by the fusion protein.

Example 54 Support of Chicken Embryo Neuron Survival

[1086] To test whether sympathetic neuronal cell viability is supportedby an albumin fusion protein of the invention, the chicken embryoneuronal survival assay of Senaldi et al may be utilized (Proc. Natl.Acad. Sci., U.S.A., 96:11458-63 (1998), which is herein incorporated byreference). Briefly, motor and sympathetic neurons are isolated fromchicken embryos, resuspended in L15 medium (with 10% FCS, glucose,sodium selenite, progesterone, conalbumin, putrescine, and insulin; LifeTechnologies, Rockville, Md.) and Dulbecco's modified Eagles medium[with 10% FCS, glutamine, penicillin, and 25 MM Hepes buffer (pH 7.2);Life Technologies, Rockville, Md.], respectively, and incubated at 37°C. in 5% CO₂ in the presence of different concentrations of the purifiedfusion protein of the invention, as well as a negative control lackingany cytokine. After 3 days, neuron survival is determined by evaluationof cellular morphology, and through the use of the calorimetric assay ofMosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)). Enhancedneuronal cell viability as compared to the controls lacking cytokine isindicative of the ability of the albumin fusion protein to enhance thesurvival of neuronal cells.

Example 55 Assay for Phosphatase Activity

[1087] The following assay may be used to assess serine/threoninephosphatase (PTPase) activity of an albumin fusion protein of theinvention.

[1088] In order to assay for serine/threonine phosphatase (PTPase)activity, assays can be utilized which are widely known to those skilledin the art. For example, the serine/threonine phosphatase (PSPase)activity of an albumin fusion protein of the invention may be measuredusing a PSPase assay kit from New England Biolabs, Inc. Myelin basicprotein (MyBP), a substrate for PSPase, is phosphorylated on serine andthreonine residues with cAMP-dependent Protein Kinase in the presence of[³²P]ATP. Protein serine/threonine phosphatase activity is thendetermined by measuring the release of inorganic phosphate from32P-labeled MyBP.

Example 56 Interaction of Serine/Threonine Phosphatases with OtherProteins

[1089] Fusion protein of the invention having serine/threoninephosphatase activity (e.g., as determined in Example 55) are useful, forexample, as research tools for the identification, characterization andpurification of additional interacting proteins or receptor proteins, orother signal transduction pathway proteins. Briefly, a labeled fusionprotein of the invention is useful as a reagent for the purification ofmolecules with which it interacts. In one embodiment of affinitypurification, an albumin fusion protein of the invention is covalentlycoupled to a chromatography column. Cell-free extract derived fromputative target cells, such as neural or liver cells, is passed over thecolumn, and molecules with appropriate affinity bind to the fusionprotein. The fusion protein-complex is recovered from the column,dissociated, and the recovered molecule subjected to N-terminal proteinsequencing. This amino acid sequence is then used to identify thecaptured molecule or to design degenerate oligonucleotide probes forcloning the relevant gene from an appropriate cDNA library.

Example 57 Assaying for Heparanase Activity

[1090] There a numerous assays known in the art that may be employed toassay for heparanase activity of an albumin fusion protein of theinvention. In one example, heparanase activity of an albumin fusionprotein of the invention, is assayed as described by Vlodavsky et al.,(Vlodavsky et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates,conditioned media, intact cells (1×10⁶ cells per 35-mm dish), cellculture supernatant, or purified fusion protein are incubated for 18 hrsat 37° C., pH 6.2-6.6, with ³⁵S-labeled ECM or soluble ECM derived peakI proteoglycans. The incubation medium is centrifuged and thesupernatant is analyzed by gel filtration on a Sepharose CL-6B column(0.9×30 cm). Fractions are eluted with PBS and their radioactivity ismeasured. Degradation fragments of heparan sulfate side chains areeluted from Sepharose 6B at 0.5<K_(av)<0.8 (peak II). Each experiment isdone at least three times. Degradation fragments corresponding to “peakII,” as described by Vlodavsky et al., is indicative of the activity ofan albumin fusion protein of the invention in cleaving heparan sulfate.

Example 58 Immobilization of Biomolecules

[1091] This example provides a method for the stabilization of analbumin fusion protein of the invention in non-host cell lipid bilayerconstucts (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999),hereby incorporated by reference in its entirety herein) which can beadapted for the study of fusion proteins of the invention in the variousfunctional assays described above. Briefly, carbohydrate-specificchemistry for biotinylation is used to confine a biotin tag to analbumin fusion protein of the invention, thus allowing uniformorientation upon immobilization. A 50 uM solution of an albumin fusionprotein of the invention in washed membranes is incubated with 20 mMNaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for1 hr at room temperature (reaction volume, 150 ul). Then the sample isdialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce ChemicalCo., Rockford Ill.) at 4C first for 5 h, exchanging the buffer aftereach hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1mM MgC]2, 10 mM sodium phosphate, pH 7). Just before addition into acuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer Rsupplemented with 50 mM octylglucoside).

Example 59 Assays for Metalloproteinase Activity

[1092] Metalloproteinases are peptide hydrolases which use metal ions,such as Zn²⁺, as the catalytic mechanism. Metalloproteinase activity ofan albumin fusion protein of the present invention can be assayedaccording to methods known in the art. The following exemplary methodsare provided:

[1093] Proteolysis of Alpha-2-Macroglobulin

[1094] To confirm protease activity, a purified fusion protein of theinvention is mixed with the substrate alpha-2-macroglobulin (0.2unit/ml; Boehringer Mannheim, Germany) in 1×assay buffer (50 mM HEPES,pH 7.5, 0.2 M NaCl, 10 mM CaC2, 25 μM ZnCl₂ and 0.05% Brij-35) andincubated at 37° C. for 1-5 days. Trypsin is used as positive control.Negative controls contain only alpha-2-macroglobulin in assay buffer.The samples are collected and boiled in SDS-PAGE sample buffercontaining 5% 2-mercaptoethanol for 5-min, then loaded onto 8%SDS-polyacrylamide gel. After electrophoresis the proteins arevisualized by silver staining. Proteolysis is evident by the appearanceof lower molecular weight bands as compared to the negative control.

[1095] Inhibition of Alpha-2-Macroglobulin Proteolysis by Inhibitors ofMetalloproteinases

[1096] Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA,AND HgCl₂), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2),and commercial small molecule MMP inhibitors) may also be used tocharacterize the proteolytic activity of an albumin fusion protein ofthe invention. Three synthetic MMP inhibitors that may be used are: MMPinhibitor I, [IC₅₀=1.0 μM against MMP-1 and MMP-8; IC₅₀=30 μM againstMMP-9; IC₅₀=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I[IC₅₀=5 μM against MMP-3], and MMP-3 inhibitor II [K₁=130 nM againstMMP-3]; inhibitors available through Calbiochem, catalog #444250,444218, and 444225, respectively). Briefly, different concentrations ofthe small molecule MMP inhibitors are mixed with a purified fusionprotein of the invention (50 μg/ml) in 22.9 μl of 1×HEPES buffer (50 mMHEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05%Brij-35)and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl ofsubstrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at37° C. for 20-hr. The reactions are stopped by adding 4×sample bufferand boiled immediately for 5 minutes. After SDS-PAGE, the protein bandsare visualized by silver stain.

[1097] Synthetic Fluorogenic Peptide Substrates Cleavage Assay

[1098] The substrate specificity for fusion proteins of the inventionwith demonstrated metalloproteinase activity may be determined usingtechniques knonw in the art, such as using synthetic fluorogenic peptidesubstrates (purchased from BACHEM Bioscience Inc). Test substratesinclude, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four areMMP substrates and the last one is a substrate of tumor necrosisfactor-α (TNF-α) converting enzyme (TACE). These substrastes arepreferably prepared in 1:1 dimethyl sulfoxide (DMSO) and water. Thestock solutions are 50-500 μM. Fluorescent assays are performed by usinga Perkin Elmer LS 50B luminescence spectrometer equipped with a constanttemperature water bath. The excitation λ is 328 nm and the emission λ is393 nm. Briefly, the assay is carried out by incubating 176 μl 1×HEPESbuffer (0.2 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 and 50 mM HEPES, pH 7.5)with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, andthen adding 20 ul of a purified fusion protein of the invention into theassay cuvett. The final concentration of substrate is 1 uM. Initialhydrolysis rates are monitored for 30-min.

[1099] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[1100] The entire disclosure of each document cited (including patents,patent applications, patent publications, journal articles, abstracts,laboratory manuals, books, or other disclosures) as well as informationavailable through Identifiers specific to databases such as GenBank,GeneSeq, or the CAS Registry, referred to in this application are hereinincorporated by reference in their entirety. The specification andsequence listing of each of the following U.S. applications are hereinincorporated by reference in their entirety: Application No. 60/229,358filed on Apr. 12, 2000; No. 60/199,384 filed on Apr. 25, 2000 and No.60/256,931 filed on Dec. 21, 2000.

1 37 1 23 DNA Artificial Sequence primer_bind primer useful to clonehuman growth hormone cDNA 1 cccaagaatt cccttatcca ggc 23 2 33 DNAArtificial Sequence primer_bind primer useful to clone human growthhormone cDNA 2 gggaagctta gaagccacag gatccctcca cag 33 3 16 DNAArtificial Sequence misc_structure synthetic oligonucleotide used tojoin DNA fragments with non-cohesive ends. 3 gataaagatt cccaac 16 4 17DNA Artificial Sequence misc_structure synthetic oligonucleotide used tojoin DNA fragments with non-cohesive ends. 4 aattgttggg aatcttt 17 5 17DNA Artificial Sequence misc_structure synthetic oligonucleotide used tojoin DNA fragments with non-cohesive ends. 5 ttaggcttat tcccaac 17 6 18DNA Artificial Sequence misc_structure synthetic oligonucleotide used tojoin DNA fragments with non-cohesive ends. 6 aattgttggg aataagcc 18 7 24PRT Artificial Sequence SITE 1)..(19) invertase leader sequence 7 MetLeu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys 1 5 10 15Ile Ser Ala Asp Ala His Lys Ser 20 8 21 DNA Artificial Sequencemisc_structure synthetic oligonucleotide used to join DNA fragments withnon-cohesive ends. 8 gagatgcaca cctgagtgag g 21 9 27 DNA ArtificialSequence misc_structure synthetic oligonucleotide used to join DNAfragments with non-cohesive ends. 9 gatcctgtgg cttcgatgca cacaaga 27 1024 DNA Artificial Sequence misc_structure synthetic oligonucleotide usedto join DNA fragments with non-cohesive ends. 10 ctcttgtgtg catcgaagccacag 24 11 30 DNA Artificial Sequence misc_structure syntheticoligonucleotide used to join DNA fragments with non-cohesive ends. 11tgtggaagag cctcagaatt tattcccaac 30 12 31 DNA Artificial Sequencemisc_structure synthetic oligonucleotide used to join DNA fragments withnon-cohesive ends. 12 aattgttggg aataaattct gaggctcttc c 31 13 47 DNAArtificial Sequence misc_structure synthetic oligonucleotide used tojoin DNA fragments with non-cohesive ends. 13 ttaggcttag gtggcggtggatccggcggt ggtggatctt tcccaac 47 14 48 DNA Artificial Sequencemisc_structure synthetic oligonucleotide used to join DNA fragments withnon-cohesive ends. 14 aattgttggg aaagatccac caccgccgga tccaccgccacctaagcc 48 15 62 DNA Artificial Sequence misc_structure syntheticoligonucleotide used to join DNA fragments with non-cohesive ends. 15ttaggcttag gcggtggtgg atctggtggc ggcggatctg gtggcggtgg atccttccca 60 ac62 16 63 DNA Artificial Sequence misc_structure syntheticoligonucleotide used to join DNA fragments with non-cohesive ends. 16aattgttggg aaggatccac cgccaccaga tccgccgcca ccagatccac caccgcctaa 60 gcc63 17 1782 DNA Homo sapiens CDS (1)..(1755) 17 gat gca cac aag agt gaggtt gct cat cgg ttt aaa gat ttg gga gaa 48 Asp Ala His Lys Ser Glu ValAla His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 gaa aat ttc aaa gcc ttggtg ttg att gcc ttt gct cag tat ctt cag 96 Glu Asn Phe Lys Ala Leu ValLeu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 cag tgt cca ttt gaa gat catgta aaa tta gtg aat gaa gta act gaa 144 Gln Cys Pro Phe Glu Asp His ValLys Leu Val Asn Glu Val Thr Glu 35 40 45 ttt gca aaa aca tgt gtt gct gatgag tca gct gaa aat tgt gac aaa 192 Phe Ala Lys Thr Cys Val Ala Asp GluSer Ala Glu Asn Cys Asp Lys 50 55 60 tca ctt cat acc ctt ttt gga gac aaatta tgc aca gtt gca act ctt 240 Ser Leu His Thr Leu Phe Gly Asp Lys LeuCys Thr Val Ala Thr Leu 65 70 75 80 cgt gaa acc tat ggt gaa atg gct gactgc tgt gca aaa caa gaa cct 288 Arg Glu Thr Tyr Gly Glu Met Ala Asp CysCys Ala Lys Gln Glu Pro 85 90 95 gag aga aat gaa tgc ttc ttg caa cac aaagat gac aac cca aac ctc 336 Glu Arg Asn Glu Cys Phe Leu Gln His Lys AspAsp Asn Pro Asn Leu 100 105 110 ccc cga ttg gtg aga cca gag gtt gat gtgatg tgc act gct ttt cat 384 Pro Arg Leu Val Arg Pro Glu Val Asp Val MetCys Thr Ala Phe His 115 120 125 gac aat gaa gag aca ttt ttg aaa aaa tactta tat gaa att gcc aga 432 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr LeuTyr Glu Ile Ala Arg 130 135 140 aga cat cct tac ttt tat gcc ccg gaa ctcctt ttc ttt gct aaa agg 480 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu LeuPhe Phe Ala Lys Arg 145 150 155 160 tat aaa gct gct ttt aca gaa tgt tgccaa gct gct gat aaa gct gcc 528 Tyr Lys Ala Ala Phe Thr Glu Cys Cys GlnAla Ala Asp Lys Ala Ala 165 170 175 tgc ctg ttg cca aag ctc gat gaa cttcgg gat gaa ggg aag gct tcg 576 Cys Leu Leu Pro Lys Leu Asp Glu Leu ArgAsp Glu Gly Lys Ala Ser 180 185 190 tct gcc aaa cag aga ctc aaa tgt gccagt ctc caa aaa ttt gga gaa 624 Ser Ala Lys Gln Arg Leu Lys Cys Ala SerLeu Gln Lys Phe Gly Glu 195 200 205 aga gct ttc aaa gca tgg gca gtg gctcgc ctg agc cag aga ttt ccc 672 Arg Ala Phe Lys Ala Trp Ala Val Ala ArgLeu Ser Gln Arg Phe Pro 210 215 220 aaa gct gag ttt gca gaa gtt tcc aagtta gtg aca gat ctt acc aaa 720 Lys Ala Glu Phe Ala Glu Val Ser Lys LeuVal Thr Asp Leu Thr Lys 225 230 235 240 gtc cac acg gaa tgc tgc cat ggagat ctg ctt gaa tgt gct gat gac 768 Val His Thr Glu Cys Cys His Gly AspLeu Leu Glu Cys Ala Asp Asp 245 250 255 agg gcg gac ctt gcc aag tat atctgt gaa aat cag gat tcg atc tcc 816 Arg Ala Asp Leu Ala Lys Tyr Ile CysGlu Asn Gln Asp Ser Ile Ser 260 265 270 agt aaa ctg aag gaa tgc tgt gaaaaa cct ctg ttg gaa aaa tcc cac 864 Ser Lys Leu Lys Glu Cys Cys Glu LysPro Leu Leu Glu Lys Ser His 275 280 285 tgc att gcc gaa gtg gaa aat gatgag atg cct gct gac ttg cct tca 912 Cys Ile Ala Glu Val Glu Asn Asp GluMet Pro Ala Asp Leu Pro Ser 290 295 300 tta gct gct gat ttt gtt gaa agtaag gat gtt tgc aaa aac tat gct 960 Leu Ala Ala Asp Phe Val Glu Ser LysAsp Val Cys Lys Asn Tyr Ala 305 310 315 320 gag gca aag gat gtc ttc ctgggc atg ttt ttg tat gaa tat gca aga 1008 Glu Ala Lys Asp Val Phe Leu GlyMet Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 agg cat cct gat tac tct gtcgtg ctg ctg ctg aga ctt gcc aag aca 1056 Arg His Pro Asp Tyr Ser Val ValLeu Leu Leu Arg Leu Ala Lys Thr 340 345 350 tat gaa acc act cta gag aagtgc tgt gcc gct gca gat cct cat gaa 1104 Tyr Glu Thr Thr Leu Glu Lys CysCys Ala Ala Ala Asp Pro His Glu 355 360 365 tgc tat gcc aaa gtg ttc gatgaa ttt aaa cct ctt gtg gaa gag cct 1152 Cys Tyr Ala Lys Val Phe Asp GluPhe Lys Pro Leu Val Glu Glu Pro 370 375 380 cag aat tta atc aaa caa aactgt gag ctt ttt gag cag ctt gga gag 1200 Gln Asn Leu Ile Lys Gln Asn CysGlu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 tac aaa ttc cag aat gcgcta tta gtt cgt tac acc aag aaa gta ccc 1248 Tyr Lys Phe Gln Asn Ala LeuLeu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 caa gtg tca act cca actctt gta gag gtc tca aga aac cta gga aaa 1296 Gln Val Ser Thr Pro Thr LeuVal Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 gtg ggc agc aaa tgt tgtaaa cat cct gaa gca aaa aga atg ccc tgt 1344 Val Gly Ser Lys Cys Cys LysHis Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 gca gaa gac tat cta tccgtg gtc ctg aac cag tta tgt gtg ttg cat 1392 Ala Glu Asp Tyr Leu Ser ValVal Leu Asn Gln Leu Cys Val Leu His 450 455 460 gag aaa acg cca gta agtgac aga gtc aca aaa tgc tgc aca gag tcc 1440 Glu Lys Thr Pro Val Ser AspArg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 ttg gtg aac agg cgacca tgc ttt tca gct ctg gaa gtc gat gaa aca 1488 Leu Val Asn Arg Arg ProCys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 tac gtt ccc aaa gagttt aat gct gaa aca ttc acc ttc cat gca gat 1536 Tyr Val Pro Lys Glu PheAsn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 ata tgc aca ctt tctgag aag gag aga caa atc aag aaa caa act gca 1584 Ile Cys Thr Leu Ser GluLys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 ctt gtt gag ctt gtgaaa cac aag ccc aag gca aca aaa gag caa ctg 1632 Leu Val Glu Leu Val LysHis Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 aaa gct gtt atg gatgat ttc gca gct ttt gta gag aag tgc tgc aag 1680 Lys Ala Val Met Asp AspPhe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 gct gac gat aaggag acc tgc ttt gcc gag gag ggt aaa aaa ctt gtt 1728 Ala Asp Asp Lys GluThr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 gct gca agt caagct gcc tta ggc tta taacatctac atttaaaagc atctcag 1782 Ala Ala Ser GlnAla Ala Leu Gly Leu 580 585 18 585 PRT Homo Sapiens 18 Asp Ala His LysSer Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn PheLys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys ProPhe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala LysThr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu HisThr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg GluThr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu ArgAsn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 ProArg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys AlaAla 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly LysAla Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln LysPhe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu SerGln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu ValThr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly AspLeu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr IleCys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys CysGlu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val GluAsn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp PheVal Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala LysAsp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg HisPro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 TyrGlu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys ValPro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn LeuGly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys ArgMet Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln LeuCys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr LysCys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe SerAla Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn AlaGlu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu LysGlu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val LysHis Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met AspAsp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp AspLys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala AlaSer Gln Ala Ala Leu Gly Leu 580 585 19 57 DNA Artificial Sequenceprimer_bind primer used to generate XhoI and ClaI site in pPPC0006 19gcctcgagaa aagagatgca cacaagagtg aggttgctca tcgatttaaa gatttgg 57 20 58DNA Artificial Sequence primer_bind primer used in generation XhoI andClaI site in pPPC0006 20 aatcgatgag caacctcact cttgtgtgca tctcttttctcgaggctcct ggaataag 58 21 24 DNA Artificial Sequence primer_bind primerused in generation XhoI and ClaI site in pPPC0006 21 tacaaacttaagagtccaat tagc 24 22 29 DNA Artificial Sequence primer_bind primer usedin generation XhoI and ClaI site in pPPC0006 22 cacttctcta gagtggtttcatatgtctt 29 23 60 DNA Artificial Sequence Misc_Structure Syntheticoligonucleotide used to alter restriction sites in pPPC0007 23aagctgcctt aggcttataa taaggcgcgc cggccggccg tttaaactaa gcttaattct 60 2460 DNA Artificial Sequence Misc_Structure Synthetic oligonucleotide usedto alter restriction sites in pPPC0007 24 agaattaagc ttagtttaaacggccggccg gcgcgcctta ttataagcct aaggcagctt 60 25 32 DNA ArtificialSequence primer_bind forward primer useful for generation of albuminfusion protein in which the albumin moiety is N-terminal of theTherapeutic Protein 25 aagctgcctt aggcttannn nnnnnnnnnn nn 32 26 51 DNAArtificial Sequence primer_bind reverse primer useful for generation ofalbumin fusion protein in which the albumin moiety is N-terminal of theTherapeutic Protein 26 gcgcgcgttt aaacggccgg ccggcgcgcc ttattannnnnnnnnnnnnn n 51 27 33 DNA Artificial Sequence forward primer useful forgeneration of albumin fusion protein in which the albumin moiety isc-terminal of the Therapeutic Protein 27 aggagcgtcg acaaaagannnnnnnnnnnn nnn 33 28 52 DNA Artificial Sequence primer_bind reverseprimer useful for generation of albumin fusion protein in which thealbumin moiety is c-terminal of the Therapeutic Protein 28 ctttaaatcgatgagcaacc tcactcttgt gtgcatcnnn nnnnnnnnnn nn 52 29 24 PRT ArtificialSequence signal signal peptide of natural human serum albumin protein 29Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 1015 Tyr Ser Arg Ser Leu Asp Lys Arg 20 30 114 DNA Artificial Sequenceprimer_bind forward primer useful for generation of PC4HSA albuminfusion VECTOR 30 tcagggatcc aagcttccgc caccatgaag tgggtaacct ttatttcccttctttttctc 60 tttagctcgg cttactcgag gggtgtgttt cgtcgagatg cacacaagagtgag 114 31 43 DNA Artificial Sequence primer_bind reverse primer usefulfor generation of PC4HSA albumin fusion VECTOR 31 gcagcggtac cgaattcggcgcgccttata agcctaaggc agc 43 32 46 DNA Artificial Sequence primer_bindforward primer useful for inserting Therapeutic protein into pC4HSAvector 32 ccgccgctcg aggggtgtgt ttcgtcgann nnnnnnnnnn nnnnnn 46 33 55DNA Artificial Sequence primer_bind reverse primer useful for insertingTherapeutic protein into pC4HSA vector 33 agtcccatcg atgagcaacctcactcttgt gtgcatcnnn nnnnnnnnnn nnnnn 55 34 17 PRT Artificial Sequencesignal Stanniocalcin signal peptide 34 Met Leu Gln Asn Ser Ala Val LeuLeu Leu Leu Val Ile Ser Ala Ser 1 5 10 15 Ala 35 22 PRT ArtificialSequence signal Synthetic signal peptide 35 Met Pro Thr Trp Ala Trp TrpLeu Phe Leu Val Leu Leu Leu Ala Leu 1 5 10 15 Trp Ala Pro Ala Arg Gly 2036 66 PRT Agkistrodon piscivorus 36 Ile Thr Tyr Thr Asp Cys Thr Glu SerGly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly LysGly Asn Lys Cys Ile Leu Gly Ser 20 25 30 Gln Gly Lys Asp Asn Gln Cys ValThr Gly Glu Gly Thr Pro Lys Pro 35 40 45 Gln Ser His Asn Gln Gly Asp PheGlu Pro Ile Pro Glu Asp Ala Tyr 50 55 60 Asp Glu 65 37 71 PRTAgkistrodon piscivorus 37 Glu Ala Gly Glu Glu Cys Asp Cys Gly Ser ProGlu Asn Pro Cys Cys 1 5 10 15 Asp Ala Ala Thr Cys Lys Leu Arg Pro GlyAla Gln Cys Ala Glu Gly 20 25 30 Leu Cys Cys Asp Gln Cys Lys Phe Met LysGlu Gly Thr Val Cys Arg 35 40 45 Ala Arg Gly Asp Asp Val Asn Asp Tyr CysAsn Gly Ile Ser Ala Gly 50 55 60 Cys Pro Arg Asn Pro Phe His 65 70

What is claimed:
 1. An albumin fusion protein comprising a memberselected from the group consisting of: (a) a Therapeutic protein:X andalbumin comprising the amino acid sequence of SEQ ID NO:18; (b) aTherapeutic protein:X and a fragment or a variant of the amino acidsequence of SEQ ID NO:18, wherein said fragment or variant has albuminactivity; (c) a Therapeutic protein:X and a fragment or a variant of theamino acid sequence of SEQ ID NO:18, wherein said fragment or varianthas albumin activity, and further wherein said albumin activity is theability to prolong the shelf life of the Therapeutic protein:X comparedto the shelf-life of the Therapeutic protein:X in an unflised state; (d)a Therapeutic protein:X and a fragment or a variant of the amino acidsequence of SEQ ID NO:18, wherein said fragment or variant has albuminactivity, and further wherein the fragment or variant comprises theamino acid sequence of amino acids 1-387 of SEQ ID NO:18; (e) a fragmentor variant of a Therapeutic protein:X and albumin comprising the aminoacid sequence of SEQ ID NO:18, wherein said fragment or variant has abiological activity of the Therapeutic protein:X; (f) a Therapeuticprotein:X, or fragment or variant thereof, and albumin, or fragment orvariant thereof, of (a) to (e), wherein the Therapeutic protein:X, orfragment or variant thereof, is fused to the N-terminus of albumin, orthe N-terminus of the fragment or variant of albumin; (g) a Therapeuticprotein:X, or fragment or variant thereof, and albumin, or fragment orvariant thereof, of (a) to (e), wherein the Therapeutic protein:X, orfragment or variant thereof, is fused to the C-terminus of albumin, orthe C-terminus of the fragment or variant of albumin; (h) a Therapeuticprotein:X, or fragment or variant thereof, and albumin, or fragment orvariant thereof, of (a) to (e), wherein the Therapeutic protein:X, orfragment or variant thereof, is fused to the N-terminus and C-terminusof albumin, or the N-terminus and the C-terminus of the fragment orvariant of albumin; (i) a Therapeutic protein:X, or fragment or variantthereof, and albumin, or fragment or variant thereof, of (a) to (e),which comprises a first Therapeutic protein:X, or fragment or variantthereof, and a second Therapeutic protein:X, or fragment or variantthereof, wherein said first Therapeutic protein:X, or fragment orvariant thereof, is different from said second Therapeutic protein:X, orfragment or variant thereof; (j) a Therapeutic protein:X, or fragment orvariant thereof, and albumin, or fragment or variant thereof, of (a) to(i), wherein the Therapeutic protein:X, or fragment or variant thereof,is separated from the albumin or the fragment or variant of albumin by alinker; and (k) a Therapeutic protein:X, or fragment or variant thereof,and albumin, or fragment or variant thereof, of (a) to (j), wherein thealbumin fusion protein has the following formula: R1-L-R2; R2-L-R1; orR1-L-R2-L-R1, and further wherein R1 is Therapeutic protein:X, orfragment or variant thereof, L is a peptide linker, and R2 is albumincomprising the amino acid sequence of SEQ ID NO:18 or a fragment orvariant of albumin.
 2. The albumin fusion protein of claim 1, whereinthe shelf-life of the albumin fusion protein is greater than theshelf-life of the Therapeutic protein:X, or fragment or variant thereof,in an unfused state.
 3. The albumin fusion protein of claim 1, whereinthe in vitro biological activity of the Therapeutic protein:X, orfragment or variant thereof, fused to albumin, or fragment or variantthereof, is greater than the in vitro biological activity of theTherapeutic protein:X, or fragment or variant thereof, in an unfusedstate.
 4. The albumin fusion protein of claim 1, wherein the in vivobiological activity of the Therapeutic protein:X, or fragment or variantthereof, fused to albumin, or fragment or variant thereof, is greaterthan the in vivo biological activity of the Therapeutic protein:X, orfragment or variant thereof, in an unfused state.
 5. An albumin fusionprotein comprising a peptide inserted into an albumin, or fragment orvariant thereof, comprising the amino acid sequence of SEQ ID NO:18 orfragment or variant thereof.
 6. An albumin fusion protein comprising apeptide inserted into an albumin, or fragment or variant thereof,comprising an amino acid sequence selected from the group consisting of:(a) amino acids 54 to 61 of SEQ ID NO:18; (b) amino acids 76 to 89 ofSEQ ID NO:18; (c) amino acids 92 tolOO of SEQ ID NO:18; (d) amino acids170 to 176 ofSEQ ID NO:18; (e) amino acids 247 to 252 of SEQ ID NO:18;(f) amino acids 266 to 277 of SEQ ID NO:18; (g) amino acids 280 to 288of SEQ ID NO:18; (h) amino acids 362 to 368 of SEQ ID NO:18; (i) aminoacids 439 to 447 of SEQ ID NO:18; (j) amino acids 462 to 475 of SEQ IDNO:18; (k) amino acids 478 to 486 of SEQ ID NO:18; and (l) amino acids560 to 566 of SEQ ID NO:18.
 7. The albumin fusion protein of claim 5,wherein said albumin fusion protein comprises a portion of albuminsufficient to prolong the shelf-life of the peptide as compared to theshelf-life of the peptide in an unfused state.
 8. The albumin fusionprotein of claim 6, wherein said albumin fusion protein comprises aportion of albumin sufficient to prolong the shelf-life of the peptideas compared to the shelf-life of the peptide in an unfused state.
 9. Thealbumin fusion protein of claim 5, wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the in vitrobiological activity of the peptide fused to albumin as compared to thein vitro biological activity of the peptide in an unfused state.
 10. Thealbumin fusion protein of claim 6, wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the in vitrobiological activity of the peptide fused to albumin as compared to thein vitro biological activity of the peptide in an unfused state.
 11. Thealbumin fusion protein of claim 5 wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the in vivobiological activity of the peptide fused to albumin compared to the invivo biological activity of the peptide in an unfused state.
 12. Thealbumin fusion protein of claim 6 wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the in vivobiological activity of the peptide fused to albumin compared to the invivo biological activity of the peptide in an unfused state.
 13. Thealbumin fusion protein of any one of claims 1-12, whi ch isnon-glycosylated.
 14. The albumin fusion protein of any one of claims1-12, which is expressed in yeast.
 15. The albumin fusion protein ofclaim 14, wherein the yeast is glycosylation deficient.
 16. The albuminfusion protein of claim 14 wherein the yeast is glycosylation andprotease deficient.
 17. The albumin fusion protein of any one of claims1-12, which is expressed by a mammalian cell.
 18. The albumin fusionprotein of any one of claims 1-12, wherein the albumin fusion protein isexpressed by a mammalian cell in culture.
 19. The albumin fusion proteinof any one of claims 1-12, wherein the albumin fusion protein furthercomprises a secretion leader sequence.
 20. A composition comprising thealbumin fusion protein of any one of claims 1-12 and a pharmaceuticallyacceptable carrier.
 21. A kit comprising the composition of claim 20.22. A method of treating a disease or disorder in a patient, comprisingthe step of administering the albumin fusion protein of any one ofclaims 1-12.
 23. The method of claim 22, wherein the disease or disordercomprises indication:Y.
 24. A method of treating a patient with adisease or disorder that is modulated by Therapeutic protein:X, orfragment or variant thereof, comprising the step of administering aneffective amount of the albumin fusion protein of any one of claims1-12.
 25. The method of claim 24, wherein the disease or disorder isindication:Y.
 26. A method of extending the shelf life of Therapeuticprotein:X, or fragment or variant thereof, comprising the step of fusingthe Therapeutic protein:X, or fragment or variant thereof, to albumin,or fragment or variant thereof, sufficient to extend the shelf-life ofthe Therapeutic protein:X, or fragment or variant thereof, compared tothe shelf-life of the Therapeutic protein:X, or fragment or variantthereof, in an unfused state.
 27. A nucleic acid molecule comprising apolynucleotide sequence encoding the albumin fusion protein of any oneof claims 1-12.
 28. A vector comprising the nucleic acid molecule ofclaim
 27. 29. A host cell comprising the nucleic acid molecule of claim28.
 30. An albumin fusion protein comprising a member selected from thegroup consisting of: (a) an interferon-alpha polypeptide and albumincomprising the amino acid sequence of SEQ ID NO:18; (b) aninterferon-alpha polypeptide and a fragment or a variant of the aminoacid sequence of SEQ ID NO:18, wherein said fragment or variant hasalbumin activity; (c) an interferon-alpha polypeptide and a fragment ora variant of the amino acid sequence of SEQ ID NO:18, wherein saidfragment or variant has albumin activity, and further wherein saidalbumin activity is the ability to prolong the shelf life of theinterferon-alpha polypeptide compared to the shelf-life of theinterferon-alpha polypeptide in an unfused state; (d) aninterferon-alpha polypeptide and a fragment or a variant of the aminoacid sequence of SEQ ID NO:18, wherein said fragment or variant hasalbumin activity, and further wherein the fragment or variant comprisesthe amino acid sequence of amino acids 1-387 of SEQ ID NO:18; (e) afragment or variant of an interferon-alpha polypeptide and albumincomprising the amino acid sequence of SEQ ID NO:18, wherein saidfragment or variant has antiviral activity or inhibits cellproliferation; (f) an interferon-alpha polypeptide, or fragment orvariant thereof, and albumin, or fragment or variant thereof, of (a) to(e), wherein the interferon-alpha polypeptide, or fragment or variantthereof, is fused to the N-terminus of albumin, or the N-terminus of thefragment or variant of albumin; (g) an interferon-alpha polypeptide, orfragment or variant thereof, and albumin, or fragment or variantthereof, of (a) to (e), wherein the interferon-alpha polypeptide, orfragment or variant thereof, is fused to the C-terminus of albumin, orthe C-terminus of the fragment or variant of albumin; (h) aninterferon-alpha polypeptide, or fragment or variant thereof, andalbumin, or fragment or variant thereof, of (a) to (e), wherein theinterferon-alpha polypeptide, or fragment or variant thereof, is fusedto the N-terminus and C-terminus of albumin, or the N-terminus and theC-terminus of the fragment or variant of albumin; (i) aninterferon-alpha polypeptide, or fragment or variant thereof, andalbumin, or fragment or variant thereof, of (a) to (e), which comprisesthe interferon-alpha polypeptide, or fragment or variant thereof, and aTherapeutic protein:X, or fragment or variant thereof, wherein saidinterferon-alpha polypeptide, or fragment or variant thereof, isdifferent from said second Therapeutic protein:X, or fragment or variantthereof; (j) an interferon-alpha polypeptide, or fragment or variantthereof, and albumin, or fragment or variant thereof, of (a) to (i),wherein the interferon-alpha polypeptide, or fragment or variantthereof, is separated from the albumin or the fragment or variant ofalbumin by a linker; and (k) an interferon-alpha polypeptide, orfragment or variant thereof, and albumin, or fragment or variantthereof, of (a) to (j), wherein the albumin fusion protein has thefollowing formula: R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, and furtherwherein R1 is interferon-alpha polypeptide, or fragment or variantthereof, L is a peptide linker, and R2 is albumin comprising the aminoacid sequence of SEQ ID NO:18 or a fragment or variant of albumin. 31.The albumin fusion protein of claim 30, wherein the shelf-life of thealbumin fusion protein is greater than the shelf-life of theinterferon-alpha polypeptide, or fragment or variant thereof, in anunfused state.
 32. The albumin fusion protein of claim 30, wherein thein vitro antiviral activity or cell proliferation inhibitory activity ofthe interferon-alpha polypeptide, or fragment or variant thereof, fusedto albumin, or fragment or variant thereof, is greater than the in vitroantiviral activity or cell proliferation inhibitory activity of theinterferon-alpha polypeptide, or fragment or variant thereof, in anunfused state.
 33. The albumin fusion protein of claim 30, wherein thein vivo antiviral activity or cell proliferation inhibitory activity ofthe interferon-alpha polypeptide, or fragment or variant thereof, fusedto albumin, or fragment or variant thereof, is greater than the in vivoantiviral activity or cell proliferation inhibitory activity of theinterferon-alpha polypeptide, or fragment or variant thereof, in anunfused state.
 34. An albumin fusion protein comprising aninterferon-alpha polypeptide inserted into an albumin, or fragment orvariant thereof, comprising the amino acid sequence of SEQ ID NO:18 orfragment or variant thereof.
 35. An albumin fusion protein comprising aninterferon-alpha polypeptide inserted into an albumin, or fragment orvariant thereof, comprising an amino acid sequence selected from thegroup consisting of: (a) amino acids 54 to 61 of SEQ ID NO:18; (b) aminoacids 76 to 89 of SEQ ID NO:18; (c) amino acids 92 to 100 of SEQ IDNO:18; (d) amino acids 170 to 176 of SEQ ID NO:18; (e) amino acids 247to 252 of SEQ ID NO:18; (f) amino acids 266 to 277 of SEQ ID NO:18; (g)amino acids 280 to 288 of SEQ ID NO:18; (h) amino acids 362 to 368 ofSEQ ID NO:18; (i) amino acids 439 to 447 of SEQ ID NO:18; (j) aminoacids 462 to 475 of SEQ ID NO:18; (k) amino acids 478 to 486 of SEQ IDNO:18; and (l) amino acids 560 to 566 of SEQ ID NO:18.
 36. The albuminfusion protein of claim 34, wherein said albumin fusion proteincomprises a portion of albumin sufficient to prolong the shelf-life ofthe interferon-alpha polypeptide as compared to the shelf-life of theinterferon-alpha polypeptide in an unfused state.
 37. The albumin fusionprotein of claim 35, wherein said albumin fusion protein comprises aportion of albumin sufficient to prolong the shelf-life of theinterferon-alpha polypeptide as compared to the shelf-life of theinterferon-alpha polypeptide in an unfused state.
 38. The albumin fusionprotein of claim 34, wherein said albumin fusion protein comprises aportion of albumin sufficient to prolong the in vitro antiviral activityor cell proliferati on inhibi tory activity of the interferon-alphapolypepti de fused to albumin as compared to the in vitro antiviralactivity or cell proliferation inhibitory activity of theinterferon-alpha polypeptide in an unfused state.
 39. The albumin fusionprotein of claim 35, wherein said albumin fusion protein comprises aportion of albumin sufficient to prolong the in vitro antiviral activityor cell proliferation inhibitory activity of the interferon-alphapolypeptide fused to albumin as compared to the in vitro antiviralactivity or cell proliferation inhibitory activity of theinterferon-alpha polypeptide in an unfused state.
 40. The albumin fusionprotein of claim 34 wherein said albumin fusion protein comprises aportion of albumin sufficient to prolong the in vivo antiviral activityor cell proliferation inhibitory activity of the interferon-alphapolypeptide fused to albumin compared to the in vivo antiviral activityor cell proliferation inhibitory activity of the interferon-alphapolypeptide in an unfused state.
 41. The albumin fusion protein of claim35 wherein said albumin fusion protein comprises a portion of albuminsufficient to prolong the in vivo antiviral activity or cellproliferation inhibitory activity of the interferon-alpha polypeptidefused to albumin compared to the in vivo antiviral activity or cellproliferation inhibitory activity of the interferon-alpha polypeptide inan unfused state.
 42. The albumin fusion protein of any one of claims30-41, which is non-glycosylated.
 43. The albumin fusion protein of anyone of claims 30-41, which is expressed in yeast.
 44. The albumin fusionprotein of claim 43, wherein the yeast is glycosylation deficient. 45.The albumin fusion protein of claim 43 wherein the yeast isglycosylation and protease deficient.
 46. The albumin fusion protein ofany one of claims 30-41, which is expressed by a mammalian cell.
 47. Thealbumin fusion protein of any one of claims 30-41, wherein the albuminfusion protein is expressed by a mammalian cell in culture.
 48. Thealbumin fusion protein of any one of claims 30-41, wherein the albuminfusion protein further comprises a secretion leader sequence.
 49. Acomposition comprising the albumin fusion protein of any one of claims30-41 and a pharmaceutically acceptable carrier.
 50. A kit comprisingthe composition of claim
 49. 51. A method of treating a disease ordisorder in a patient, comprising the step of administering the albuminfusion protein of any one of claims 30-41.
 52. The method of claim 51,wherein the disease or disorder comprises a member selected from thegroup consisting of: Hairy cell leukemia; Kaposi's sarcoma; genitalwarts; anal warts; chronic hepatitis B; chronic non-A, non-B hepatitis;hepatitis C; hepatitis D; chronic myelogenous leukemia; renal cellcarcinoma; bladder carcinoma; ovarian carcinoma; cervical carcinoma;skin cancer; recurrent respirator papillomatosis; non-Hodgkin'slymphoma; cutaneous T-cell lymphoma; melanoma; multiple myeloma; AIDS;multiple sclerosis; and glioblastoma.
 53. A method of treating a patientwith a disease or disorder that is modulated by interferon-alpha, orfragment or variant thereof, comprising the step of administering aneffective amount of the albumin fusion protein of any one of claims30-41.
 54. The method of claim 53, wherein the disease or disordercomprises a member selected from the group consisting of: Hairy cellleukemia; Kaposi's sarcoma; genital warts; anal warts; chronic hepatitisB; chronic non-A, non-B hepatitis; hepatitis C; hepatitis D; chronicmyelogenous leukemia; renal cell carcinoma; bladder carcinoma; ovariancarcinoma; cervical carcinoma; skin cancer; recurrent respiratorpapillomatosis; non-Hodgkin's lymphoma; cutaneous T-cell lymphoma;melanoma; multiple myeloma; AIDS; multiple sclerosis; and glioblastoma.55. A method of extending the shelf life of interferon-alpha, orfragment or variant thereof, comprising the step of fusing theinterferon-alpha, or fragment or variant thereof, to albumin, orfragment or variant thereof, sufficient to extend the shelf-life of theinterferon-alpha, or fragment or variant thereof, compared to theshelf-life of the interferon-alpha, or fragment or variant thereof, inan unfused state.
 56. A nucleic acid molecule comprising apolynucleotide sequence encoding the albumin fusion protein of any oneof claims 30-41.
 57. A vector comprising the nucleic acid molecule ofclaim
 56. 58. A host cell comprising the nucleic acid molecule of claim57.
 59. An albumin fusion protein comprising albumin, or a fragment orvariant thereof, and a protein selected from the group consisting of:(a) serum cholinesterase; (b) alpha-I antitrypsin; (c) aprotinin; (d)coagulation complex; (e) von Willebrand factor; (f) fibrinogen; (g)factor VII; (h) factor VIIA activated factor; (i) factor VIII; (j)factor IX; (k) factor X; (l) factor XIII; (m) cl inactivator; (n)antithrombin III; (o) thrombin; (p) prothrombin; (q) apo-lipoprotein;(r) c-reactive protein; (s) protein C; and (t) immunoglobulin.
 60. Analbumin fusion protein comprising a single chain antibody or portionthereof and albumin comprising the amino acid sequence of SEQ ID NO:18or fragment or variant thereof.